Method of dietary treatment for genetic and epigenetic diseases and disorders

ABSTRACT

Genetic and epigenetic diseases and disorders are treated with FDA approved dietary compositions in the form of dietary supplements and nutraceuticals. The diseases and disorders to be treated include Rett syndrome, trinucleotide repeat diseases such as Fragile X syndrome, memory impairment, chronic inflammation, pre-cancerous conditions which involve cancer stem cells, and post-cancerous conditions which involve cancer stem cells which have survived in spite of cancer treatment. Treatment options include prophylactic treatment which is initiated prior to the development of the symptoms of the disease or disorder.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.15/950,877, filed Apr. 11, 2018, which is divisional application of U.S.Ser. No. 15/251,880, filed Aug. 30, 2016, now U.S. Pat. No. 9,968,580,which claims priority to U.S. provisional application No. 62/283,462,filed Sep. 2, 2015, the disclosures of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to the therapeutic use of dietary supplements andnutraceuticals which have an epigenetic effect on gene expression.Various diseases and disorders involve inappropriate levels of geneexpression which can be corrected epigenetically. Examples include RettSyndrome, various trinucleotide repeat diseases, and cancerous andpre-cancerous cells in which tumor suppressor genes that have beenepigenetically silenced. The use of dietary supplements andnutraceuticals which have an epigenetic effect can beneficially improvethe gene expression profile for those who have these diseases anddisorders.

BACKGROUND OF THE INVENTION 1. Definitions, Glossary, and Abbreviations

Administration of a compound: Causing a compound to enter into the bodyof an animal, either orally, by injection, or by any other means.

Chromatin: An organized structure of the DNA (and its attached RNA andproteins) of a single chromosome that controls the compactness andaccessibility of the DNA.

CpG dinucleotide (CpG): A pair of nucleotides within a strand of DNAconsisting of a cytosine followed by a guanine in the forward readingdirection of the DNA strand. Due to C:G and G:C base paring, each CpGdinucleotide on one strand will be base paired with a (reverse reading)CpG island on the other “antisense” strand of double stranded DNA (whichis read in the reverse direction from the “sense” strand that codes forproteins).

CpG island: A region of DNA where the density of CpG dinucleotides ishigh. A typical CpG island could be 1000 nucleotides long, with a CpGdinucleotide every 10 nucleotides (on average). A CpG dinucleotide isconsidered to be methylated if most (e.g. 80%) of its CpGs aremethylated, or demethylated if few (e.g. 20%) of its CpGs aremethylated.

CpG methylation: A CpG where the cytosine is methylated (forming5-methyl cytosine).

Dietary supplement [FDA definition]: A dietary supplement is a producttaken by mouth that contains a “dietary ingredient” intended tosupplement the diet. The “dietary ingredients” in these products mayinclude vitamins, minerals, herbs or other botanicals, amino acids, andsubstances such as enzymes, organ tissues, glandulars, and metabolites.Dietary supplements can also be extracts or concentrates, and may befound in many forms such as tablets, capsules, softgels, gelcaps,liquids, or powders. They can also be in other forms such as a bar, butif they are, information on their label must not represent the productas a conventional food or a sole item of a meal or diet.

DNA Methyltransferase (DNMT): An enzyme that binds to DNA and canmethylate a CpG site. DNMT1 is considered to be the key maintenancemethyltransferase in mammals (e.g. keeping CpG islands methylated thatwere already methylated). DNMT3 enzymes can methylate CpG sites thatwere not already methylated (de novo methylation). In practice they areboth involved in establishing and maintaining the methylation state ofCpG sites (e.g. in the absence of DNMT1 or DMNT3, demethylation canoccur).

Ectopic: In an abnormal place or position. For example, if a specificCpG island on a particular type of cell's DNA is not normallysignificantly methylated, a cell of that type in which that CpG islandis significantly methylated would be said to have an ectopicallymethylated CpG island.

Ectopic acetylation: Acetylation in a place or position that would notnormally be acetylated.

Ectopic deacetylation: Deacetylation in a place or position that wouldnormally be acetylated.

Ectopic methylation: Methylation in a place or position that would notnormally be methylated.

Ectopic demethylation: Demethylation in a place or position that wouldnormally be methylated.

Epigenetics: The study of cellular and physiological phenotypic traitvariations that are caused by heritable DNA modifications that switchgenes on and off and affect how cells read genes, instead of beingcaused by changes in the DNA sequence itself. Epigenetics literallymeans “above” or “on top of” genetics. Epigenetic DNA modificationstypically involve attachments to the DNA, for example a methyl groupattached to a cytosine nucleotide, or a nucleosome that has the DNAwound upon it.

Epigenetic marker: A general term for the state of a site associatedwith gene expression that can have more than one state, resulting inmore or less gene expression as a result of the state of the marker. Forexample, a lysine on a specific histone affecting the expression of agene on a chromosome in a cell may have the alternatives of acetylation,methylation, or no modification at all. These are three alternativesstates for that epigenetic marker.

Epigenetic pattern: The pattern of epigenetic markers that results in apattern of gene expression for that cell. Each type of cell will have aset of epigenetic patterns that are appropriate for that type of cell.

FCC Grade: The Food Chemicals Codex (FCC) is a compendium of standardsused internationally for the quality and purity of food ingredients likepreservatives, flavorings, colorings and nutrients. FCC gradeingredients are approved for use in foods, dietary supplements, andcosmetics.

Food additive: A compound that is listed in the “Everything Added toFoods in the United States (EAFUS)” FDA database.

Fortify (food): To increase the nutritive value of food, especially withmicronutrients.

Histone acetylation (HAc): The attachment of an acetyl group to a lysinelocated on the amino-terminal tail of a histone. The shorthand code“H3K9Ac” indicates that Lysine (K) 9 of histone 3 is acetylated.

Histone acetyl transferase (HAT): An enzyme that acetylates histones,typically at their amino-tail lysine residues. The acetyl group donorfor HAT enzymes is Acetyl-CoA.

Histone Deacetylase Inhibitor (HDACi): A compound that inhibits theactivity of a histone deacetylase enzyme, thereby increasing theacetylation of histones. Histone deacetylase activity is sometimesinferred from the observation of increased histone acetylation, whichdoes not necessarily distinguish between true HDACi activity, theenhancement of histone acetyl transferase activity or even thenon-enzymatic acetylation of histones. Some HDACis are known to bothinhibit histone deacetylases and enhance histone acetyl transferaseactivity, thereby increasing net histone acetylation by both mechanisms.

Histone methylation (HMe): The attachment of a methyl group to a lysinelocated on the amino-terminal tail of a histone. The shorthand code“H3K27Me” indicates that Lysine (K) 27 of histone 3 is methylated.

Metabolism: The entire set of chemical reactions that can occur within aliving organism. This includes anabolism (the formation of more complexmolecules from simple ones), catabolism (the breakdown of complexmolecules from complex molecules to make simpler ones) and also simplerreactions, such as thiol-disulfide exchange reactions.

Metastable: Stable provided that it is subjected to no more than smalldisturbances, and capable of being so long-lived as to be stable forpractical purposes.

Micronutrient: A chemical element or substance that is required by aliving organism in minute amounts for normal growth.

Mitigate: To make less severe or less intense.

Non-coding RNA (ncRNA): Any RNA molecule that is produced by DNAtranscription but does not code for a protein. ncRNAs have a variety offunctions, resulting in a variety of function-specific names such assiRNA, microRNA (miRNA), long, non-coding RNA (IncRNA), etc.

Nucleosome: A structure formed from 8 histone proteins (2 each of H2A,H2B, H3, and H4 histones) on top of which DNA can be wound in order toprovide chromatin compaction and thereby control the accessibility ofgenes for transcription.

Nutraceutical: A food containing health-giving food additives and havingmedicinal benefit.

Nutrigenomics: The scientific study of the interaction of nutrition andgenes, especially with regard to the prevention or treatment of disease.

Prodrug: An inert compound that becomes active for its purpose onlyafter it is transformed or metabolized by the body.

Prophylactic treatment: Preventative treatment in order to avoid thedevelopment of a disease or condition.

Therapeutic Window: The dosage range from the minimum beneficial dosageto the maximum tolerable dosage.

Treatment: The willful administration of a therapeutic agent with theintent of preventing or mitigating a disease or disorder.

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3. Description of the Published Art

3.1 New Knowledge About Genetic Diseases

Modern methods greatly increase the amount of physiological detail whichcan be measured or observed, in some cases causing revisions tolong-held beliefs. This is especially true now that genetic informationis available for the entire coding regions of the human genome.

Diseases associated with gross genetic defects have been known fordecades, but only a small minority of the population inherit anyspecific genetic defect which, in and of itself, produces a disease.Most genetic diseases are now believed to be associated with acombination of genetic alleles or mutations (none of which individuallycause the disease), or the combination of one or more genetic factorswith environmental factors (either environmental exposure or “lifestyle”choices).

Although appropriate lifestyle choices have long been recognized asbeing key to achieving and maintaining wellness (e.g. cessation ofsmoking, avoiding drug or alcohol abuse, adequate exercise, a properdiet, . . . ), for individuals these are frequently easier said thandone.

Even though there is general agreement as to the importance of a properdiet, there are still disagreements about what a proper diet consistsof. Major ongoing controversies regarding the benefits or risks ofdietary choices include carbohydrates vs. fats, vegetarianism, veganism,vitamin supplements, added sugars, high fructose corn syrup, organicfood, pesticides, non-GMO, etc.

Lost in the noise is the importance of micronutrients.

The emerging field of nutragenomics focuses on the interaction ofnutrition and genes, especially with regard to the prevention ortreatment of disease. The relationship goes both ways. Proper geneexpression depends upon proper nutrition. But proper nutrition for anindividual depends in part upon that individual's genome.

Twin studies provide a means for determining the interplay between genesand the environment in the development of specific diseases. Monozygotic(identical) twins share almost 100% of their genes, which means thatmany of their characteristics will be nearly the same, and any majordifferences are likely to be due to differences in environmentalexposure or experience. But dizygotic (fraternal) twins share only about50% of their genes.

The classical twin study design compares the degree of twin similarityfor some characteristic, both for a set of monozygotic twins and a setof dizygotic twins. If the monozygotic twins are considerably moresimilar than the dizygotic twins (as is the case for most traits), thisis strong evidence that genes play an important role for that trait. Andif monozygotic twins show a strong divergence for a particular trait,this is evidence that the environment plays an important role for thattrait.

3.2 Rett Syndrome, a Well-Studied Genetic Disease

Although Rett syndrome (RTT) is a rare disease (occurring inapproximately 1 in 10,000 live female births), it is a devastatingdisease which typically produces severe mental and physical retardation.The affected girls typically have no verbal skills and about 50% ofaffected individuals cannot walk.

Infant development is normal until about 6 to 18 months of age,including the learning of basic skills such as purposeful hand use,development of gross motor skills such as crawling or walking, and earlylanguage development. After the onset of disease, learning progressessentially stops and these skills are progressively lost. Repetitivestereotyped hand movements, such as wringing and/or repeatedly puttinghands into the mouth, also develop.

In 90% of the cases, the cause of Rett syndrome is mutation of the MeCP2gene on the X chromosome, which codes for the MeCP2 (Methyl CpG bindingProtein 2) protein. Approximately 95% of the time the mutation is denovo (e.g. a sporadic mutation from the father that originated duringspermatogenesis), although about 5% of the time it is inherited from themother.

There are strong motivations for trying to develop therapies for Rettsyndrome:

1. The cause is well known.

2. The disease has been extensively researched.

3. There are multiple mouse models that replicate the disease.

4. There are a variety of treatments that have been tested (mostly usingeither mouse models in vivo or mouse cells in vitro) and have shown somebeneficial results.

5. There is evidence (from the mouse models) that the disease may bereversible, even years after it developed.

6. The patients with Rett syndrome require almost continuous care (theycan't feed themselves, they shouldn't be left in one position for toolong, they cannot dress themselves, etc.), and their parents aredesperate for an effective treatment to be developed.

3.2.1 the MeCP2 Gene is X-Linked

Although females have two copies of the MeCP2 gene (one on each Xchromosome), males only have one copy. Females with RTT almost alwayshave one defective copy of MeCP2 (mutated) and one good copy (wildtype), but the disease causing allele is dominant. Because males onlyhave one copy, if it is mutated the disease is more severe than infemales, and for human males it is almost always lethal by the age oftwo years.

Due to X-inactivation, only one of the female's X chromosomes is fullyactive, resulting in females having nearly the same level of Xchromosome gene products as males (known as “gene dosage”). Early in thedevelopment of the female fetus, each of its cells randomly inactivateseither the maternal or the paternal X chromosome by epigeneticallysilencing (approximately ¾) of its genes (by hypermethylating the CpGislands in the promoter regions of each of these genes). Roughly half ofthese cells will have inactivated the maternal X chromosome, and theothers will have inactivated the paternal X chromosome, although due torandomness the split may be somewhat skewed (e.g. 60/40). Becauseepigenetic programming is inherited when cells divide, all of thedaughter cells derived from each of these cells will inactivate thatsame chromosome. Because the fetus had multiple cells by the timex-inactivation occurs, different tissue samples from an individual canhave different skews.

Because monozygotic twins can have different X-inactivation patterns(and skews), resulting in a different proportion of cells expressingdefective MeCP2, the results of a study of two monozygotic twins withdiscordant Rett syndrome Kunio, 2013) are interesting. Monozygotic(identical) twins have been widely used in genetic studies to determinethe relative contributions of heredity and the environment in humandiseases. In this case, although they have the same heredity, their Xchromosome inactivation pattern is also part of their “nature”, andtherefore it was of interest to see if they had significantly differentX-inactivation skews (otherwise, their difference in disease severitywould be attributed to environmental effects).

From Kunio, 2013:

“Twin 1 (RS1), who has a milder phenotype, developed normally until 2.5years of age; she was able to use a spoon, to run and jump, and to climbstairs. At age 2, she communicated using two-word sentences. At 2.5years she started to lose learned words and the ability to communicate .. . . At 3 years and 5 months, she lost purposeful hand skills andstarted to exhibit stereotypical hand movements . . . . At age 12 years,she had generalized convulsions and her EEG showed epileptic discharges.Since then, an antiepileptic drug has been administered and her seizuresare well controlled. At age 13 years, she could run and jump, was ratherhyperactive and slimmer than twin 2, was able to reach for and graspobjects, liked to swim and to watch children's TV programs.”

“Twin 2 (RS2), who has a more severe phenotype, . . . Her developmentduring the first 6 months appeared normal but she soon started to lag.She was able to hold her head steady at 6 months, could roll over at 9months, and could sit by herself at 9 months. She had marked hypertoniaand never walked. At age 12 months, she spoke using simple words, suchas “momma” and “dada”, and could grasp a toy, but she lost theseabilities later and started to exhibit stereotypical hand movements . .. . At 2 years and one month, she had afebrile seizures, and her EEGshowed eleptiform spike discharges. At 6 years of age, an antiepilepticdrug was administered. At age 7 years, she was unable to stand, walk, orcommunicate with others. At age 13 years she (still) could not stand andrequired a wheelchair.”

. . .

“In the present study, we examined the genome, epigenome and expressionpatterns of MZ twins discordant for RTT. We found that (1) the twinsshared the same de novo MeCP2 mutation; (2) the de novo mutation was ofpaternal origin (occurred in spermatogenesis); (3) XCI (X Chromosomeinactivation) did not differ in various peripheral tissues between thetwins; (4) no inter-twin difference was found in whole gene sequences;(5) there were no differences in DNA methylation of the MeCP2 promoterregion, nor did MeCP2 expression differ between the twins; (6) the DNAmethylation status of a number of loci varied between the twins; (7)this DNA methylation difference was confirmed by the effect onexpression of three genes, which may contribute to clinical differencesbetween the twins. These results indicate that epigenetic differences,but not genetic differences, appear to be associated with thediscordance between these twins.” [Kunio, 2013]

FIG. 4 of the paper [Kunio, 2013] dramatically illustrates the resultsof a genome wide analysis of DNA methylation in the twins. Over 60 genesare shown in a red vs. green color scheme, with most of the genes (˜⅔)being more methylated (redder) for the more severely afflicted twin(RS2) and the other ⅓ being less methylated (greener). The epigeneticdifferences are truly profound, even though there was no differencefound between their X-inactivation patterns.

Other studies comparing the X-inactivation patterns of monozygotic twinshave also found no significant relationship between X-inactivationskewing and RTT disease severity, confirming that the discordanceobserved between twins cannot be explained by X-inactivation skewing.For example, in a study of two pairs of sisters with RTT, where eachpair of sisters had the same MeCP2 mutation and balanced X-inactivation,one individual from each pair could not speak or walk, and had aprofound intellectual deficit, while the other individual could speakand walk and had only moderate intellectual disability [Grillo, 2013].

Similarly, a study of a mother (healthy) in which the mutant allele waspredominantly active (75% vs. 25% in peripheral leukocytes) and herdaughter with the identical mutation who developed severe RTT concludedthat the presence of non-random XCI in the peripheral blood cells didnot provide an explanation for the normal phenotype of the carriermother Ohinata, 2008). The daughter's symptoms were described as:

-   -   “Early motor development of the patient was normal; she was able        to hold her head at 4 months and could sit unaided at 7 months.        Development abnormalities were first noted at the age of 10        months when she showed inconsolable crying and stopped smiling.        The parents also noted that her face lacked expression. At 11        months of age, she lost her ability to sit unaided and became        less interested in her toys. Physical examination showed        hypotonia, strabismus, and intention tremor of her upper limbs .        . . . At 13 months of age, she developed her first seizures        characterized by tonic movement of the upper limbs and loss of        consciousness. At the time, electroencephalography and brain        magnetic resonance did not reveal any abnormalities. Thus, she        was diagnosed with Rett syndrome, which was further confirmed by        a mutation on the MeCP2 gene. At this point, the parents        requested genetic testing to assess the risk of having more        affected children.”

Skewed X chromosome inactivation in the brain was investigated in nineRTT brains (obtained from the Harvard Brain Tissue Resource Center)[Gibson, 2004]. Balanced XCI patterns were observed in allneuroanatomical regions examined. They concluded that blood is morelikely to undergo skewing than neural tissues in RTT patients.

In mouse models of RTT, male mice survive birth and develop the RTTphenotype at an earlier age than female mice, so most experiments areperformed using male mice.

In RTT patients, the level of expression of MeCP2 is typically normal(as measured by the mRNA level), even though any MeCP2 protein that isproduced is defective. Interestingly, there is another disease (MeCP2duplication syndrome) where the over expression of MeCP2 producesRTT-like symptoms in both humans [Na, 2013] and mice [Na, 2012].

3.3 What Is Known About MeCP2 Function?MeCP2 is a protein which wasalready known to bind to DNA at a methylated “CpG site”, which is alocation on the DNA where a cytosine nucleotide is immediately followedby a guanine nucleotide, and the cytosine has a methyl group attached tocarbon #5 (“5-methyl Cytosine”). This can have the effect of inhibitingthe transcription of the following gene. MeCP2 is found in all cells ofthe body, but its most important functions seem to be in neurons. Theassociation between MeCP2 mutation and Rett syndrome was discovered in1999 [Amir, 1999], which greatly increased interest in the(mis)functions of this protein.

By 2001 it was discovered that deficiency of MeCP2 in CNS neuronsresults in a Rett-like phenotype in mice [Chen, 2001]. From then on,most experimental research on RTT has used mouse models of the disease.3.3.1 Early Research Focuses on BDNF

In 2003 it was discovered that MeCP2 can control the level of BDNF (the“Brain Derived Neurotrophic Factor” protein) [Chen, 2003]. BDNF isinvolved in promoting neurite growth and synapse formation (learning)and in their preservation (memory).

In 2007 it was shown that restoration of MeCP2 function in adult mice(by activating the transgene expression of a functional MeCP2 gene)partially reverses the disease [Guy, 2007]:

“Our study shows that RTT-like neurological defects due to the absenceof the MeCP2 gene can be rectified by delayed restoration of that gene.The experiments do not suggest an immediate therapeutic approach to RTT,but they establish the principle of reversibility in a mouse model and,therefore, raise the possibility that neurological defects seen in thisand related human disorders are not irrevocable.”

In 2009, treatment of MeCP2 mutant mice with “Insulin-like Growth Factor1” (IGF-1) partially rescued 9 separate measures of RTT symptoms inmice: “(i) lifespan, (ii) locomotor activity, (iii) respiratoryfunction, (iv) heart rate, (v) brain weight, (vi) concentration of apostsynaptic density protein in the motor cortex, (vii) spine density onmotor cortex neurons, and (ix) cortical circuit plasticity.” [Tropea,2009]. Perhaps what is most impressive is the number of separatemeasures in which they were able to show improvement. Theirjustification for IGF-1 treatment was “Like BDNF, IGF-1 is widelyexpressed in the CNS during normal development . . . , strongly promotesneuronal cell survival and synaptic maturation . . . , and facilitatesthe maturation of functional plasticity in the developing cortex.”

In 2010 it was shown in a mouse model of RTT that Valproic acidtreatment increased BDNF and also normalized (made more similar to wildtype) the levels of various proteins in neuroblastoma cells [Vecsler,2010]. Furthermore, exogenous BDNF was shown to normalize the synapticfunction of mouse brainstem slices [Kline, 2010], and glatiramer acetatetreatment was shown to increase BDNF in a mouse model of RTT [Ben-Zeev,2011].

Although the above treatments may make it seem that treating RTT onlyrequires increasing BDNF, there are a multitude of genes whoseexpression is affected in RTT. Just increasing BDNF only partiallyimproves the RTT phenotype. Other treatments have been found that seemto be more effective.

Table 1 below attempts to list all of the pharmaceutical treatments thathave shown beneficial results in animal models of RTT (and also thetests that have been performed in humans). The list is probablyincomplete, but it should be representative.

TABLE 1 # Disease Model Treatment Outcome First author, year 1 Humanfemales L-Carnitine Improved Patient Well Being Index, no Ellaway, 1999effect on Hand Apraxia Scale assessment 2 MeCP21^(lox) mice Choline ↑Dark-cycle locomotor activity, ↑ Motor Nag, 2007 function 3MeCP2^(tm1.1Jae) mice Ampakine Normalization of breathing, ↑ BDNF Ogier,2007 4 MeCP2^(tm1.1Bird) mice Desipramine ↓ Apneas, ↑ TH expressingneurons Roux, 2007 5 MecP2-null mice IGF-1 ↑ Lifespan, ↑ Normalbreathing, ↑ Motor Trpoea, 2009 function 6 MeCP2 knockdown in Valproicacid Normalization of proteins in Vecsler, 2010 human SK-NSH cellsneuroblastima cells: ↑ MeCP2, ↑ BDNF, ↑ AcH3 7 MeCP2^(tm1.1Jae) miceExogenous BDNF Normalized synaptic function in Kline, 2010 brainstemslices 8 MeCP2^(tm1 HzO) mice Glatiramer acetate ↑ BDNF Ben-Zeev, 2011 9MeCP2^(tm1.1Jae) mice 7,8- ↑ Lifespan, ↑ Normal breathing, ↑ MotorJohnson, 2011 dihydroxyflavone function 10 MeCP2^(−ly) mice Fingolimod ↑Lifespan, ↓ Hind-limb clasping, ↑ Motor Deogracias, 2012 function 11MeCP21^(lox) mice Acetyl-L-Carnitine ↑ Forepaw grip strength, ↑ Motorfunction Schaevitz, 2012 12 MeCP2^(tm1.1Jae) mice Ketamine Improved PPIof the ASR (cognitive Kron, 2012 function) 13 Human females ω-3 PUFA ↑Hand use, ↑ Nonverbal communication, De Felice, 2012 ↑ Motor function,Normalized breathing 14 MeCP2^(tm1.1Bird/+) mice Fluvastatin Delayedsymptoms, ↑ Lifespan, ↑ Motor Buchovecky, 2013 function 15 Human femalesTopiramate Improved neurological symptoms, ↑ Krajnc, 2013 Motility 16Human females ω-3 PUFA Normalized gene expression in brain De Felice,2013 17 MeCP2 KO mice Triheptanoin ↓ Apneias Park, 2014 18MeCP2^(tm1.1Jae) mice Valproic Acid ↑ Lifespan, ↓ Hind-limb clasping, ↑Motor Guo, 2014 function, ↓ Tremor 19 Human females IGF-1 ↑ THexpressing neurons Khwaja, 2014 20 MeCP2^(tm1.1Bird) mice LevodopaInflammation (isoprostanes), ↑ Bone Szczesna, 2014 density 21 Humanfemales ω-3 PUFA Protects microtubules from Signorini, 2014depolymerization (and improves the transport of BDNF in neurons) 22MeCP2-mutated human Tubastatin A ↑ dendrite growth Gold, 2015fibroblasts 23 Silenced MeCP2 in mouse Pentabarbatol ↑ Synaptictransmission (calcium spikes) Ma, 2015 neurons Abbreviations: ↑increased, ↓ decreased

Although 4 of the 8 treatments that were tested up to 2011 (numbers 1-8in the list) involved increased BDNF, the evidence from Chen, 2003(above), which I described as “MeCP2 can control the level of BDNF”actually showed that MeCP2 normally depresses the expression of the BDNFgene, and only increases the BDNF protein level when there is excesscalcium in the neuron (e.g. after the neuron has “fired”). MeCP2 isdescribed as “a selective regulator of neuronal gene expression.Activity-dependent transcription underlies the ability of the nervoussystem to convert the effects of transient stimuli into long-termchanges in brain function” (i.e. learning and memory)[Chen, 2003].

3.3.2 Later Research Investigates a Broad Range of Treatments

The 16 remaining treatments listed in Table 1 use 14 differentpharmacological agents, and although they all show some benefit(otherwise they wouldn't be in the table), two stand out: (1) The threeexperiments using Omega-3 polyunsaturated fatty acids (“ω-3 PUFA”) wereconducted using human females with RTT, and showed definite benefits forthe patients (improvements in hand use, nonverbal communication, motorfunction, and breathing) [De Felice, 2012] and in biomarkers forinflammation [De Felice, 2013; Signorini, 2014]. (2) The experimentswith Fluvastatin treatment using mice which delayed disease symptomdevelopment and improved motor function and lifespan [Buchovecky, 2013].

3.3.2.1 Treatment with ω-3 PUFA

This research group had previously established that a variety of markersof oxidative stress are elevated in RTT patients [De Felice, 2009].Oxidative stress markers included intraerythrocyte non-protein-boundiron (NPBI; i.e., free iron, plasma NPBI, F₂-isoprostanes (F₂-IsoPs, asfree, esterified, and total forms), and protein carbonyls. Markers ofoxidative stress were significantly increased in RTT subjects:intraerythrocyte NPBI (2.73 fold, “×2.73”), plasma NPBI (×6.0), freeF₂-IsoP (×1.85), esterified F₂-IsoP (×1.69), total F₂-IsoP (×1.66), andprotein carbonyls (×4.76).

Based upon this evidence of enhanced oxidative stress and lipidperoxidation in RTT patients, they tested the possible therapeuticeffects ω-3 PUFAs on the clinical symptoms and oxidative stressbiomarkers in the earliest stage of RTT. The treatment group (treatedwith fish oil, which is high in ω-3 PUFAs) and the control group eachhad 20 patients, and the study duration was 6 months. In the treatmentgroup, significant improvements were observed for motor/independentsitting, ambulation, hands use, non-verbal communication, andrespiratory dysfunction, while a non-significant trend was observed forlanguage [De Felice, 2012]. Using the RTT Clinical Severity Score (CSS)[Neul, 2008] to evaluate the patients, the untreated group had their CSSincrease from 37 to 39 (range 0-58), while the treatment group had theirCSS improve from 37 down to 21. A short video clip made from parents'home movies is available at:link.springer.com/article/10.1007%2Fs12263-012-0285-7.

Secondary outcomes (measurements of oxidative stress) include a markeddecrease in plasma F₂-dihomo-IsoPs (−86.3%), F₃-IsoPs (−55.15%), NPBI(−42.2%), F₄-NeuroPs (−40.3%), and intraerythrocyte-NPBI (−46.3%). Nosignificant improvement in any of the examined oxidative stress markerswas observed in the untreated group.

Another study by the same group measured the blood plasma proteomeprofile with untreated RTT, after treatment with ω-3 PUFAs as fish oilfor 12 months, and healthy controls [De Felice, 2013]. Sixteen proteinswere found to be significantly differentially expressed in the RTTpatients (compared to controls). In untreated patients, 10 of theproteins upregulated in the range of +1.17 to +2.07 fold and 6 of theproteins were downregulated in the range of −1.32 to −2.56 fold (Table 1of De Felice, 2014). After 12 months of treatment, the proteinexpressions of the RTT patients were normalized with the previouslyupregulated now being in the range of +1.26 to −1.03 fold, while theprotein expressions that were downregulated now being in the range of+1.33 to +1.03 fold compared to controls (calculated from Table 1 of DeFelice, 2013).

Recent work by the same group has confirmed that all mouse models forRTT that have been tested show evidence of oxidative stress and lipidperoxidation (increased plasma NPBI, F₂—IsoPs and F₂-dihomo-IsoPs). Andbrain-specific MeCP2 gene reactivation fully rescues brain oxidativestress, returning to the level of age-matched wild type litter-mates [DeFelice, 2014].

3.3.2.2 Treatment with Fluvastatin

A genetic screen for suppressors of symptoms of RTT in a MeCP2 mousemodel was used to try to identify pathways that are responsible fordisease pathology. They raised 679 MeCP2^(tm1.1Bird)/y mice most ofwhich had severe enough neurological abnormalities that they had eitheralready died or had to be euthanized by 6-16 weeks of age; however, someof the mice showed amelioration of one or more health assessment traits.Further selection followed by genetic mapping identified a nonsensemutation of the Sqle gene (encoding the enzyme squalene monooxidase)which is part of the pathway for cholesterol synthesis. [Buchovecky,2003]

They then hypothesized that the heterozygous Sqle mutation ameliorates apreviously unrecognized dysregulation of cholesterol metabolism inMeCP2-null mice. They reasoned that a pharmacologic inhibitor ofcholesterol synthesis might produce an attenuation of symptomscomparable to that of the genetic inhibitor (mutated Sqle gene) inMeCP2-null mice.

Fluvastatin (or Lovastatin, which was also successfully tested) cancross the blood-brain barrier, and therefore is suitable for use intreating a neurological disorder. Fluvastatin treated MeCP2-null miceshowed improved rotarod performance (motor function, 200 seconds time tofall versus 60 seconds for MeCP2-null controls) and increased longevity(no deaths during the 270 day experiment versus 30% mortality for theMeCP2-null controls). Lipid profiles (serum cholesterol, total liverlipids) were somewhat normalized by the Fluvastatin treatment (closer tothe values for wild type controls).

Caveats are that they did not evaluate (or at least didn't report) theeffects on other abnormalities known to be associated with RTT. Also the“combined health score” (limbclasping, tremors, and activity)[Guy, 2007]got worse with successive Fluvastatin treatments (from 5 to 10treatments), especially when the dosage was increased, to the pointwhere there was no significant benefit from the Fluvastatin treatment ifthe dosage was the 10× dose [Buchovecky, 2003, Supplementary FIG. 10].

The loss of effectiveness when the Fluvastatin dosage is increased maybe due to the inhibition of synthesis of gerangygeranoil that occurswith statin treatment (which inhibits cholesterol synthesis at the HMGCRenzyme, see FIG. 2). But the synthesis of gerangygeranoil is probablyincreased in the Sqle mutant mouse, due to the loss of Sqle activity.Therefore, statin treatment diverges from the mutant Sqle mouse model inthis respect, perhaps with great significance.

-   -   “Cholesterol turnover is also required to produce        gerangygeranoil, a product of HMGCR upstream of SQLE that is        essential for learning and synaptic plasticity, and is important        for the interaction between neurons and astrocytes at the        synapse.” [Buchovecky, 2003]

The loss of effectiveness may also be due to cholesterol synthesis beingessential for synapse development [Suzuki, 2007] as well as forgerangygeranoil synthesis [Kotti, 2006]. Perhaps the lower dose ofFluvastatin preserves enough cholesterol synthesis to allow synapses tobe formed.

In summary, various treatments have shown benefits in mouse models ofRett syndrome. But except for treatment with ω-3 PUFAs, apparently therehas not been a clear enough perception of benefit (or of safety) toinduce clinicians (or their Institutional Review Boards) to move on tohuman clinical trials, even for treatments that have already beenapproved for use in clinical trials involving children (e.g. thesuccessful treatment with Fluvastatin for children and adolescents withheterozygous familial hypercholesterolaemia [van der Graff, 2006]).

3.4 Further Evidence that RTT is an Epigenetic Disease

3.4.1 A Brief Introduction to Epigenetics

There is more to genetics than the DNA sequence that determines thegenes. Not only are genes themselves inherited, the expression patternsfor the genes is inherited as well. This is true both for the individualchild and also for the cells within the child, where daughter cellsinherit their gene expression patterns whenever a cell divides.

Commonly, a daughter cell will inherit a gene expression pattern whichis different from the gene expression pattern of its parent cell. Theparent cell may be a pluripotent stem cell and its daughter cells mayinclude tissue-specific stem cells. And their daughter cells may befurther differentiated to perform their specific functions. All of thesecells share the same DNA sequence, but their DNA has been epigeneticallymodified in order to restrict the genes that are expressed within eachspecific type of cell after differentiation.

For example, a bone marrow stem cell (e.g. a hemocytoblast) can haveeither a “common myeloid progenitor” cell or a “common lymphoidprogenitor” cell as a daughter cell. The common myeloid progenitor cellcan in turn have either a megakaryocite, an erythrocyte, a mast cell ora myeoblast as a daughter cell. Of these, only the erythrocyte and themast cell are fully differentiated (the other cell types can havedaughter cells that are more further differentiated).

One way that the gene expression of daughter cells is restricted is bythe methylation of specific nucleotides in the DNA (the cytosines of CpGsites), especially at CpG islands within the promotor region for thegenes. The addition of a methyl groups to cytosine nucleotides (cytosinemethylation) has the effect of reducing gene expression and has beenfound in the cells of every vertebrate examined. When DNA is beingcopied for cell division, the methylation pattern is copied as well, sothat (in the absence of differentiation) the gene expression pattern forthe daughter cell will start out as that of its parent. Or themethylation pattern will change in a cell-specific way when producing adaughter cell that is differentiated from its parent.

During the life of a cell, the methylation pattern can change. There isconstant “methylase” and “demethylase” enzyme activity, which tends tomaintain the existing pattern, but can respond to intranuclear (andultimately to extracellular) signaling as well. However, for anyspecific type of cell the methylation pattern of the DNA should remainrelatively constant (especially in the “CpG islands” in the promotorregions for genes), because this is what keeps the cell-type constant.But even when the methylation pattern remains constant, there iscontinuous turnover of the methylation at individual CpG sites withinmethylated CpG islands. The methyltransferase enzymes (especially DNMTI)will remethylate a CpG within a methylated CpG island in order to keepthe CpG island methylated as a whole, even in the presence of variationsin the methylation of individual CpG sites within the CpG island.

Another way that the gene expression of cells is epigeneticallycontrolled is by controlling the access of transcription factors to theDNA. Small lengths of DNA are wound on top of “nucleosome” structuresthat are composed of “histone” proteins. If a section of DNA is tightlywound on top of a nucleosome, it is unavailable to transcription factorsand the expression of its associated gene will be limited. DNA that isfree (not attached to nucleosomes, or at least temporarily released), ismore available to transcription factors and its associated gene is morelikely to be expressed.

Both the location of nucleosomes on the DNA and tightness of DNAattachment is controlled by specialized proteins that can modify thehistones. In particular, there are “histone acetyltransferase” (HAT)enzymes that can attach acetyl groups to the “tails” of histones, withthe effect of loosening the winding of DNA on the nucleosome. And thereare “histone deacetylase” (HDAC) enzymes that can remove acetyl groupsfrom the histone tails. And there are “histone deacetylase inhibitor”(HDACi) molecules that can prevent the HDAC proteins from deacetylatingthe histones, thereby keeping an acetylated histone acetylated and theDNA attached to its nucleosome accessible for transcription.

There are actually a wide variety of modifications that occur on histonetails (e.g. they can also be methylated, phosphorylated, or ubiquinatedat various positions). Histone modifications occur more often thanmodifications to the DNA methylation pattern, and can fine-tune theoperation of the cell. But there is some cross-talk in both ways betweenhistone modifications and DNA methylation, which helps preserve thestability of the cell-type.

3.4.2 Shared Histone Modifications Across Various RTT Patients

Most studies of RTT involve only human females (or mouse males) thathave a mutated MeCP2 gene. But one study involving humans included RTTpatients whose MeCP2 genes were both functional [Kauffman, 2005]. Of the17 patients with RTT, 11 had MeCP2 mutations (“RTTPos”) and 7 did not(“RTTNeg”). There were also 10 gender-matched controls.

Among the patients with MeCP2 mutations, three had the R270X truncationmutation, two had the R168X truncation mutation, and one of each had theDe1796 deletion, R255X truncation, V288X truncation, R306C missense,T158M missense, and R294X truncation mutations. A reasonably diverseset.

The presence (HAc+) or absence (HAc−) of histone acetylation wasdetermined by immunochemistry in lymphocytes for histone H3 and forhistone H4. Averages for the 3 groups (Control, RTTPos and RTTNeg) wereas follows:

H3Ac+ H3Ac− H3Ac+/H3Ac− Control 851.7 936.4 3.69 ± 2.14 RTTPos 1030.6379.1 0.52 ± 0.21 RTTNeg 2045.7 863.5 0.44 ± 0.11 H4Ac+ H4Ac−H4Ac+/H4Ac− Control 820.6 648.9 0.96 ± 0.33 RTTPos 266.4 422.4 3.01 ±1.06 RTTNeg 476.8 584.8 2.39 ± 1.55

Amazingly, all of the Controls can be distinguished from all of the RTTpatients by these simple measurements, regardless of the patient's typeof MeCP2 mutation, or even when MeCP2 is not mutated at all. Forexample, the lowest H3Ac+/H3Ac− for any control was 1.55 (3.69−2.14) butthe highest H3Ac+/H3Ac− for any patient was 0.73 (0.52+0.21). Similarly,the H4Ac+/H4Ac− values for the Controls were widely separated from theH4Ac+/H4Ac− of the patients.

In further experiments, they determined that for the specific lysineH3K9 the ratio of acetylation to non-acetylation for controls was 5.02but for RTTPos it was 0.54 and for RTTNeg it was 0.32. Similarly, forthe specific lysine H3K14, the ratio of acetylation to non-acetylationfor controls was 2.90 but for RTTPos it was 0.82 and for RTTNeg it was0.96.

In further experiments, they determined that for the specific lysineH3K4 the degree of methylation for controls was 0.145 but for RTTPos itwas 0.023 and for RTTNeg it was 0.014. Similarly, for the specificlysine H3K9, the degree of methylation for controls was 0.538 but forRTTPos it was 0.054 and for RTTNeg it was 0.046.

It should be noted that H3K9Ac, H3K14Ac, H3K4Me and H3K9Me are allpositively related to gene expression, so their relatively high valuesin Controls compared to patients indicates that lymphocyte genes arebeing expressed in Controls that are not being expressed in the RTTpatients, regardless of the type of MeCP2 mutation or even whether MeCP2is mutated at all.

This supports the observation by Kunio quoted above that even in twinswith an identical MeCP2 mutation, “These results indicate thatepigenetic differences, but not genetic differences, appear to beassociated with the discordance between these twins.”

In other words, RTT can be viewed to be primarily an epigenetic diseasewhich has MeCP2 mutations as a significant risk factor.

SUMMARY OF THE INVENTION 4. Summary of the Invention

There are various diseases and disorders which have inappropriate geneexpression in cells, which is largely under epigenetic control. Forexample: (1) cellular differentiation involves the silencing of geneswhose expression is inappropriate for that specific type of cell, whileenabling the expression of the genes that are appropriate to be (atleast sometimes) expressed, (2) beyond the silencing of genes, theexpression of genes in each cell can also be fine-tuned by epigeneticcontrol, (3) because the epigenetic state of gene silencing in a cell isheritable, an inappropriate epigenetic pattern of gene silencing in onecell can be inherited as the default epigenetic pattern of genesilencing for its daughter cells.

Although most epigenetic markers are heritable, they remain changeable,and may even be changed as a result of dietary input. However, there isan inherent stability for every specific type of cell which causes theepigenetic pattern to tend towards a pattern that is appropriate forthat type of cell.

In other words, ectopic (inappropriate for that location, for that typeof cell) epigenetic markers are metastable (not permanently stable, butnot necessarily changing any time soon), while genetic markers that areappropriate for that cell type are inherently stable (but may none theless be modifiable).

Particular types of cells may have multiple epigenetic patterns that areappropriate for that cell type, with the ability to switch between a setof alternative patterns. This can happen according to the developmentalstage of the individual. Each of these patterns can be stable, requiringan external input to switch them to the next appropriate pattern.

The present invention uses dietary ingredients (e.g. in a nutraceuticalor a dietary supplement) to modify the epigenetic pattern of some cellsin the body so that they will transition to appropriate epigeneticpatterns that alleviate a disease or disorder.

Specifically, dietary ingredients that provide bioavailable acetate canincrease the acetylation of proteins (such as histone proteins) andother biomolecules with a net effect on gene expression profiles ofcells, disrupting their dysfunctional epigenetic patterns and causingthem to adopt more appropriate patterns for the treatment of thesediseases and disorders.

In an aspect, the present disclosure encompasses a method of treating agenetic or epigenetic disease or disorder. The method comprises theadministration to an animal a therapeutically effective dose of acomposition containing acetate selected from the group of calciumacetate, magnesium acetate, sodium acetate, potassium acetate,ethylacetate, or any combination thereof, said composition formingacetate in the body and said acetate increasing the level of proteinacetylation in the subject. The amount of acetate administered per daymay be about 100 mg to about 15,000 mg of acetate per day. In variousembodiments, the amount of acetate may also be about 100 mg to about5000 mg per day, about 200 mg to about 2000 mg per day, or about 500 mgto about 1000 mg per day. In various other embodiments, the amount ofacetate may be about 375 mg to about 15,000 mg per day, about 750 mg toabout 7500 mg per day, about 750 mg to about 5000 mg per day, or about750 mg to about 3000 mg per day. In certain embodiments, the compositionis a nutraceutical or a dietary supplement.

In another aspect, the present disclosure encompasses a method fortreating Rett Syndrome in a subject in need thereof. The methodcomprises administering to the subject about 100 mg to about 15,000 mgof acetate per day, wherein the acetate is selected from calciumacetate, magnesium acetate, sodium acetate, potassium acetate,ethylacetate, or any combination thereof. In various embodiments, theamount of acetate may also be about 100 mg to about 5000 mg per day,about 200 mg to about 2000 mg per day, or about 500 mg to about 1000 mgper day. In various other embodiments, the amount of acetate may beabout 375 mg to about 15,000 mg per day, about 750 mg to about 7500 mgper day, about 750 mg to about 5000 mg per day, or about 750 mg to about3000 mg per day. The daily amount of acetate may be formulated as acomposition to be administered in one or doses. In certain embodiments,the composition may be a nutraceutical or a dietary supplement.Administration of the acetate may increase protein (including but notlimited to histones) acetylation in blood lymphocytes as well as inother cell types, increase synaptic function, increase synapticformation, increase expression of BDNF, improve memory, improveslearning, and/or improve motor function. Methods for measuring theseeffects are known in the art.

In another aspect, the present disclosure encompasses a method fortreating cancer in a subject in need thereof. The method comprisesadministering to the subject about 100 mg to about 15,000 mg of acetateper day, wherein the acetate is selected from calcium acetate, magnesiumacetate, sodium acetate, potassium acetate, ethylacetate, or anycombination thereof. In various embodiments, the amount of acetate mayalso be about 100 mg to about 5000 mg per day, about 200 mg to about2000 mg per day, or about 500 mg to about 1000 mg per day. In variousother embodiments, the amount of acetate may be about 375 mg to about15,000 mg per day, about 750 mg to about 7500 mg per day, about 750 mgto about 5000 mg per day, or about 750 mg to about 3000 mg per day. Thedaily amount of acetate may be formulated as a composition to beadministered in one or doses. In certain embodiments, the compositionmay be a nutraceutical or a dietary supplement. Administration of theacetate may increase protein acetylation (including, but not limited to,histone acetylation), inhibit proliferation of cancer cells, inhibittumor growth, reduce a tumor's size, decrease methylation at CpG islandsin cancerous cells in the subject, and/or increase expression of one ormore tumor suppressor genes. Methods for measuring these effects areknown in the art.

In another aspect, the present disclosure encompasses a method fortreating a trinucleotide repeat disorder in a subject in need thereof.The method comprises administering to the subject about 100 mg to about15,000 mg of acetate per day, wherein the acetate is selected fromcalcium acetate, magnesium acetate, sodium acetate, potassium acetate,ethylacetate, or any combination thereof. In various embodiments, theamount of acetate may also be about 100 mg to about 5000 mg per day,about 200 mg to about 2000 mg per day, or about 500 mg to about 1000 mgper day. In various other embodiments, the amount of acetate may beabout 375 mg to about 15,000 mg per day, about 750 mg to about 7500 mgper day, about 750 mg to about 5000 mg per day, or about 750 mg to about3000 mg per day. The daily amount of acetate may be formulated as acomposition to be administered in one or doses. In certain embodiments,the composition may be a nutraceutical or a dietary supplement.Administration of the acetate may increase protein acetylation(including, but not limited to, histone acetylation), inhibitproliferation of cancer cells, inhibit tumor growth, reduce a tumor'ssize, and/or decrease methylation at CpG islands in the subject. Methodsfor measuring these effects are known in the art.

Other aspects and iterations of the invention are described morethoroughly below

BRIEF DESCRIPTION OF THE DRAWINGS 5. Brief Description of the Drawings

The application file contains at least one photograph executed in color.Copies of this patent application publication with color photographswill be provided by the Office upon request and payment of the necessaryfee.

FIG. 1 shows various molecules of interest. (FIG. 1A) Acetyl group;(FIG. 1B) Acetylated lysine; (FIG. 1C) Acetyl-CoA; (FIG. 1D) AceticAcid; (FIG. 1E) Butyrate; (FIG. 1F) ω-3 Fatty Acid; (FIG. 1G) ValproicAcid; (FIG. 1H) Triheptanoin.

FIG. 2 (derived from “Squalene_pathway_map.png”; Wikipedia) shows therole of the SQLE enzyme in cholesterol synthesis.

FIG. 3 (From Weng, 2011) shows how the RTT symptom score varies with agefor MeCP2 null mice.

FIG. 4 (from Madiraju, 2009) shows that Acetylcarnitine can enter thecell nucleus and be converted to Acetyl-CoA.

FIG. 5 (from Boffa, 1978) shows the inhibition of histone deacetylaseenzyme activity by butyrate treatment.

FIG. 6 (from Cousens, 1979) shows the HDACi activity of various shortchain fatty acids.

FIG. 7 (from Hinnebusch, 2002) shows the histone hyperacetylation fromtreatment with various short chain fatty acids.

FIG. 8 (from Gold, 2015) shows the high HDACi activity of Tubastatin A.

FIG. 9 (from Chapkin, 2008) Synergy between ω-3 and butyrate treatment.

FIG. 10 shows more molecules of interest. (FIG. 10A) Sodium Acetate;(FIG. 10B) Potassium Acetate; (FIG. 10C) Dichloroacetate; (FIG. 10D)Magnesium Acetate; (FIG. 10E) Calcium Acetate; (FIG. 10F) GlycerinTriacetate.

FIG. 11 (from Ma, 2007) shows that p53-null cells use glycolysis for ATPproduction.

FIG. 12 (from vater, 2015) shows genes affected by mutation in PCNSL.

FIG. 13 (from Detich, 2003) shows that valproic acid demethylates CpGsites.

FIG. 14 (from Condorelli, 2008) shows cell viability after HDACitreatments.

FIG. 15 Metabolic Products of Dichloroacetate.

FIG. 16 (from Biacsi, 2008) shows gene reactivation from HDACitreatment.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

6.1 Pharmacological Treatments for RTT Revisited

Treatment with Valproic acid shows up twice in the listing of beneficialtreatments (Table 1), with the benefits of: (1) normalizing proteinlevels, (2) increased MeCP2 expression (e.g. from the good gene), (3)increased BDNF, (4) increased histone H3 acetylation, (5) improvedneurological symptoms, (6) improved motility, and (6) normalized geneexpression in the brain. Valproic acid is well known as a histonedeacetylase inhibitor (HDACi), which increases the acetylation ofhistones, and this is recognized as its principal pharmacologicalactivity.

Other treatments listed in Table 1 which have HDACi activity as theirprincipal pharmacological activity are Topiramate and Tubastatin A. Iwas curious which other of the treatments have known histone acetylationactivity (in addition to whatever other activities they have). Theresults of this survey are listed in Table 2 below, which includes abrief description of the histone acetylation mechanism and a referencethat provides more details. Clearly, the vast majority of beneficialpharmacological treatments have at least the side effect of increasinghistone acetylation. (Even some of the other beneficial treatments mayhave histone acetylation as an undocumented side effect, but I wasn'table to find a reference to document it.) The list of treatments isdiverse, but their methods of action may not be as diverse as it wouldotherwise seem.

TABLE 2 # Disease Model Treatment ↑ HAc? Histone acetylation Firstauthor, year 1 Human females L-Carnitine Y Increases Acetyl-CoAMadiraju, 2009 2 MeCP21^(lox) mice Choline N 3 MeCP2^(tm1.1Jae) miceAmpakine N 4 MeCP2^(tm1.1Bird) mice Desipramine N 5 MecP2-null miceIGF-1 Y Increases H3, H4 Histone acetylation Sun, 2006 in the brain 6MeCP2 knockdown in Valproic acid Y HDACi Eyal, 2004 human SK-NSH cells 7MeCP2^(tm1.1Jae) mice Exogenous BDNF N 8 MeCP2^(tm1 HzO) mice Glatirameracetate Y Contains acetate 9 MeCP2^(tm1.1Jae) mice 7,8- Ndihydroxyflavone 10 MeCP2^(−ly) mice Fingolimod Y HDACi Hait, 2014 11MeCP21^(lox) mice Acetyl-L-Carnitine Y Increases Acetyl-CoA Madiraju,2009 12 MeCP2^(tm1.1Jae) mice Ketamine Y HDACi activity in the NucleusReus, 2013 Accumbens region of the brain 13 Human females ω-3 PUFA YIncrease the HDACi activity of Xu, 2006 butyrate 14 MeCP2^(tm1.1Bird/+)mice Fluvastatin Y Inhibits HDAC activity and induces Lin, 2008 histoneacetylation 15 Human females Topiramate Y HDACi Eyal, 2004 16 Humanfemales ω-3 PUFA Y Increases the HDACi activity of Xu, 2006 butyrate 17MeCP2 KO mice Triheptanoin Y Metabolizes to Acetyl-CoA Park, 2014 18MeCP2^(tm1.1Jae) mice Valproic Acid Y HDACi Eyal, 2004 19 Human femalesIGF-1 Y Increases H3, H4 Histone acetylation Sun, 2006 in the brain 20MeCP2^(tm1.1Bird) mice Levodopa Y Histone acetylation, via Inden, 2012phosphorylation 21 Human females ω-3 PUFA Y Increases the HDACi activityof Xu, 2006 butyrate 22 MeCP2-mutated Tubastatin A Y HDACi Gold, 2015human fibroblasts 23 Silenced MeCP2 in Pentabarbatol N mouse neurons

6.2 the Development of RTT Revisited.

Infants with RTT develop normally up to an age of 6 to 18 months(disease onset) including their ability to learn. This age correspondsto the developmental stage where the MeCP2 protein level in neuronsincreases in normal (non-RTT) infants and brain glucose metabolismincreases [Shahbazian, 2002]. Infants with RTT may either develop anormal increase in MeCP2 (mutant, non-functional) protein, or themutation (e.g. a deletion) may prevent the MeCP2 from being produced atall. In either case, disease symptoms first appear at the developmentalstage where MeCP2 should start performing its function. Withoutfunctioning MeCP2, learning seems to stop, and previously learned skillsare gradually lost.

-   -   “[In RTT] the regression phase is best explained by a delayed        onset of neuronal dysfunction.” [Shahbazian, 2002]

Normal infants actually change the way that they learn at about this age(e.g. when they reach this developmental stage). According to Liston,2002:

-   -   “Infants of 6 months old can remember events for up to 24 hours,        which extends to up to a month when they are 9 months old . . .        . In humans, the brain undergoes important changes towards the        end of the first year, including the growth and differentiation        of dendrites in the hippocampus, which continue into the second        year. These developmental processes should increase the        efficiency of integration and registration of information in the        neocortex, and in the prefrontal cortex in particular . . . .    -   [Their experiments show that before this developmental        transition infants need repeated exposure to something in order        to learn it (they learn slowly), but that after this transition        they can remember something that has happened only once.]

. . . our results support the popular belief that at 9 months thehippocampus and regions of the frontal cortex are not yet fully mature.They also indicate that there is a neurobiological component to memoryenhancement across the second year, contrary to early assumptions thatthis is entirely attributable to experience.”

In other words, at this stage of development neurons change the way theywork, and instead of using repetition to develop synaptic connections,they can turn on a “synapse formation machine” in response to a singleexperience.

It seems that for infants with RTT, at this stage of development, theold way of learning gets turned off and the new way gets turned on. Butthe new way doesn't work.

Interestingly, the same thing happens in RTT mice. For both humans andmice, there is an increase in total MeCP2 protein as neuronsdifferentiate [Dragich, 2007], but the mouse models allow more detailedanalysis because brain tissue samples can be analyzed for any age ofmouse. There are two splice variants for the MeCP2 gene, MeCP2e1 andMeCP2e2. At about 21 days of age (week 3), the distribution profilechanges with MeCP2e2 becoming predominantly localized to the dorsalthalamus and cortical layer V [Dragich, 2007], indicating that theneurons in these regions are further differentiated than neurons inother regions of the brain. This is when (at week 4) symptoms of RTTfirst become evident in the mice. See FIG. 3, “RTT Symptom Score”, [fromFIG. 1C of Weng, 2011].

6.3 Function of MeCP2 Revisited

The main function of MeCP2 in neurons is to produce “switchable on oroff” chromatin condensation which when switched off can simultaneouslyenable the expression of a whole set of genes at one time. In effect,this turns on or off a “synapse formation machine”. The MeCP2 proteinhas a binding site that is specific for methylated CpG (e.g. a DNAlocation) at one end of the protein and an “A:T hook” at the other end(e.g. it specifically binds to a DNA location that has a high density ofA and T nucleotides). Along the length of MeCP2, it may be attached tothe DNA (causing “DNA compaction” and preventing the expression ofgenes) or the MeCP2 can separate from that section of the DNA. Whetheror not this stretch of DNA is attached to the MeCP2 depends upon theabsence or presence of calcium, which enters the neuron when the neuronfires. [This is a simplified description of a multi-step process. Formore details, see Tao, 2009]

In effect, when the neuron fires, this stretch of DNA becomes availablefor gene transcription. The BDNF gene is one of these genes, so theexpression of BDNF (and hence the production of BDNF protein) getsswitched on in response to calcium [Chen, 2003; Zhaolan, 2006].

In the absence of MeCP2 (and even in the presence of MeCP2), BNDFexpression requires activation of its promoters. BDNF is a neurotrophin(promotes nerve growth, synapse formation, and synapse maintenance)which is produced by neurons, secreted from vesicles, and sensed bycells that have BDNF receptors on their surface. So BDNF does much ofthe signaling involved in the cooperative synapse formation betweenneurons, and is probably involved in early infant learning (fromrepetitive experience) as well as in MeCP2 dependent learning (after theassociated developmental stage).

What may be needed is a way to turn back on the early method oflearning, even though the RTT patient has gotten old enough that thismode of learning has been turned off.

6.4 Epigenetics Revisited

The mapping from genotype to phenotype for every cell in the body iscontrolled by epigenetics. We are gradually learning the mechanismsinvolved, which include at least: (1) DNA modification (CpG islandmethylation, CpG site methylation, chromatin condensation, histoneattachment, histone positioning, MeCP2 attachment, RNA attachment,transcription factor attachment . . . ), (2) histone modification(acetylation, methylation, phosphorylation, ubiquination, . . . ), (3)various forms of RNA (including siRNA, non-coding RNA, long non-codingRNA, and micro RNA). Note that the production of any particular RNA fromthe DNA can be controlled by gene expression, controlled by pseudo geneexpression, or the RNA may be produced merely because the “junk DNA” isbeing transcribed.

Although in general the phenotype of any individual cell remains stable,the epigenetic program for a cell can be modified in exceptionalcircumstances. For example, when a cell divides, the expression ofspecific transcription factors can produce different phenotypes for ofeach of the daughter cells.

-   -   “During the differentiation process, the developmental capacity        of totipotent cells in the early embryo is progressively lost as        these undertake cell fate decisions. This process is driven by        the expression of cross-antagonistic transcription factors (TF)        promoting development towards one cell fate while repressing an        alternative differentiation path. Cell fate decisions are        fortified by progressive acquisition of epigenetic modifications        at both the DNA and chromatin level. While cell identity is        undeniably dictated by the expression profile guided by cell        type-specific TFs, the robustness of the acquired        transcriptional state is crucially dependent upon the        configuration of the chromatin context in which these TFs        operate . . . .    -   . . . In a model whereby TF cross-antagonism is the central        mechanism by which cell fate is determined, cell fate        transitions, such as those observed during de-differentiation        and trans-differentiation events, are possible through ectopic        expression of the required cell type instructive TFs.” [Nashun,        2015]

With regards to RTT, it is clear that neurons have two“differentiations”, one for “repeated experience” based learning andanother for “one shot” learning from a single experience. What may beneeded for RTT patients is a method for switching the differentiationstate of the neuron back, in order to re-enable the “repeatedexperience” mode of learning for the patient. To do this we need toepigenetically transition the cell to its previous neuronal phenotype.

-   -   “Developmental progression from a totipotent to a differentiated        cell is a gradual process accompanied by deposition of        repressive histone marks and by increasing compaction.” [Nashun,        2015]

But going back to a previous differentiation state involves making thehistones less restrictive (which will naturally make the DNA lesscompact). In other words, we need to increase the histone acetylation.(It is not clear at this stage whether we can permanently change thestate with one treatment, or whether we will need to continue thetreatment for life. The assumption is that if we stop the treatment, theneuron could transition to the more “developed” phenotype. Therefore, weshould expect to treat the RTT patient for life, and the treatmentshould have very low (or no) toxicity.)

6.5 Why Aren't Epigenetic Treatments Highly Toxic?

Epigenetic treatments are generally not highly toxic because there aremultiple epigenetic feedback mechanisms to sustain the stability of thecellular phenotype. Although the expression of each individual gene islargely controlled by the combination of its own promoters' methylationand its histones' acetylation/methylation state, it also responds to theRNA that is transcribed by other genes (and pseudo genes) which tend toreinforce the persistence of the cellular phenotype.

In other words, if the expression of an individual gene becomesdisturbed in a way that could change the cell's phenotype, it will tendto be corrected soon due to the combined effect of the pattern of thevarious RNAs that were already produced (based upon the existingphenotype), as described below. It is only those genes that do notchange the cell's phenotype that are freely responsive to the signalingenvironment of the cell.

6.5.1 X-Inactivation Revisited

The stability of X-inactivation illustrates the role of RNA inovercoming perturbations to the cell's methylation/acetylationsignaling. In an experimental system, the impact of separatelyperturbing each of the epigenetic aspects involved (methylation,acetylation, and Xist RNA synthesis) showed that none of these could,when individually manipulated, switch off the X-inactivation of the cell[Csankovszki, 2001]. Autofluorescence was used to detect X-inactivationand 100,000 to 500,000 cells were analyzed for each bulk sample.

The inactive X chromosome (Xi) expressed Xist, a nuclear untranslatedRNA that coats that chromosome. Once an X chromosome is inactivated, theinactive state of the chromosome is clonally inherited through manyrounds of cell division. To study the effect of deletion of Xist fromXi, a conditional allele of the gene Xist was introduced onto thechromosome (a “conditional mutant”). After X-inactivation wasestablished, switching off Xist had minimal impact (only a few cellslost their X-inactivation, and only for a short duration because afteranother week in culture even these cells had spontaneously recoveredtheir X-inactivation). Even with Xist expression continuously switchedoff for >2 months, allowing the cells to go through many rounds of celldivision, the cells' X-inactivation persisted.

Because DNA methylation and hypoacetylation of core histones arebelieved to contribute to the inactivation of X-linked genes, they alsodid experiments to test the effects of 5-aza-2′-deoxycytidine (5-azadC)treatment (for DNA demethylation, a well-known use of 5-azadC) orTrichostatin A (TSA) treatment (TSA is a well-known HDACi).

After allowing conditionally mutant cells to go through several roundsof cell division, half of the cultures were treated with TSA (to inhibithistone deacetylase activity and thereby increase histone acetylation).This made no change in the X-inactivation of the cells, even if Xist wasturned off.

The separate effect of DNA demethylation was tested in cultures treatedwith 5-azadC. A small number of cells lost their X-inactivation (19× asmany compared to controls). Combining Xist deletion with DNAdemethylation increased the number of cells that lost theirX-inactivation (30× as many compared to controls).

Combining 5-azadC and TSA treatment (with Xist on) caused a small numberof cells to lose their X-inactivation (60× as many compared tocontrols).

By comparison, a conditional Xist/Dnmt1 double mutant, when it had bothmutations turned on, had an increase of 2500× in the number of cellsthat lost their X-inactivation relative to controls.

Their conclusion was that “Xist RNA, histone deacetylation, and DNAdemethylation act synergistically to achieve extraordinary stability ofX chromosome silencing”. If any of the three mechanisms is left intact,“reactivation rates [were] comparable to mutation rates” [Csankovszki,2001].

6.5.2 Ectopic Xist Gene Activation is not Stable

Another group has studied the effect of attempting to do X-inactivationin a human male cell line. They developed a transgenetic conditionalcell line that allows Xist gene expression to be turned on at will (eventhough Xist expression would never normally be turned on in a male wildtype cell). Turning on Xist produces X-inactivation in this cell line,but not to the same degree as is observed in female cells (“the size ofthe [inactivation region] was less that is observed for the inactive Xchromosome in female somatic cells”) [Chow, 2007].

They observed that although some of the epigenetic marks for genesilencing were present (e.g. histone 4 lysine 20 is methylated), andthat these marks were Xist dependent. Turning off Xist resulted ingradual re-expression of genes that had been silenced (e.g. EGFP whichcoded for a green fluorescent protein). After 30 days, the epigeneticmarks for this gene had returned to their pre-Xist levels. [Chow, 2007]

It is apparent that although X-inactivation is incredibly stable incells where it is appropriate (e.g. in female cells), ectopic geneactivation (activation where it is not appropriate) is relativelyunstable. This is because only appropriate gene expression is supportedby a cell phenotype dependent feedback network that resists geneexpression that is inconsistent with the differentiation-dependent celltype. Every legitimate type of cell has a feedback network to preserveits phenotype (and to only allow it to adopt another phenotype if itreceives the specific signals that induce its differentiation into anappropriate type of daughter cell).

6.5.3 Non-Coding RNAs Mediate Orchestral Regulation of the Cell

6.5.3.1 RNA-Mediated Transcriptional Gene Silencing

RNA interference (RNAi) is a process whereby a small double-strandedinterfering RNA (siRNA) molecules functionally target and direct thedegradation of a homology containing mRNA (this technique has been knownsince 1999) [Hamilton, 1999]. It has more recently been discovered thatsmall single stranded non-coding RNAs can bind to DNA in a sequencedependent manner (using part of the length of the ncRNA strand) whilerecruiting an “epigenetic remodeling” protein complex to that DNAlocation (using another portion of the ncRNA strand), ultimatelyresulting in the epigenetic remodeling of the target site to atranscriptionally silent state [Morris, 2011].

Some micro RNAs (miRNAs, a form of small RNA) function to directtranscriptional gene silencing in human cells by causing the methylationof histone 3 lysines 9 and 27, leading to DNA CpG island methylation atthe targeted promoter. These targeted epigenetic changes appearspecifically at the RNA target site and are not found at distalun-targeted regions, suggesting a level of specificity [Morris, 2009].

-   -   “The current mechanistic understanding of [transcriptional gene        silencing] is that within the first 24 hours following small RNA        treatment there is a robust increase in Argonaute 1 [a protein        involved in epigenetic remodeling] at the targeted promoter        followed shortly thereafter by increasing concentrations of H3K9        dimethylation and H3K27 trimethylation . . . suggesting that        small RNA guides an epigenetic remodeling complex to a        particular target loci. When the small RNA targeting is        sustained for 3-4 days, DNA methylation [at the CpG island]        begins to appear and correlates with the observation of longterm        stable gene silencing.” [Morris, 2009]

Any gene, in addition to coding for its protein, can have non-coding RNAtranscribed when it is activated by a specific promoter, and these ncRNAcan silence another gene (or maybe a bunch of genes). And thisdownstream gene (when activated by a specific promoter) can express itsown ncRNAs to silence other genes (or even to activate the originalgene, see below). Because a gene can have several associated promoters,and the ncRNAs that it responds to (and the ncRNAs that it produces) canbe based upon its epigenetic phenotype, a signaling network is formedthat is cell-type specific and can serve to maintain its phenotypicidentity.

There are also “pseudo genes” which do not code for any protein, butthey have promotor regions that can cause the gene to express variousncRNAs to silence other genes [Weinberg, 2013].

An interesting example of this comes from the pseudogene Tsix (Xistspelled backwards, which turns out to be appropriate), which codes anuntranslated RNA that is antisense and complementary for Xist, the RNAthat is essential for the X-inactivation of one of the female Xchromosomes [Chow, 2005]. When Tsix is expressed, the resulting RNAattaches to any Xist in the area, forming a double stranded RNA, andthereby inactivating the Xist RNA. Because Tsix RNA is formed locally tothe active X-chromosome, this prevents the active X-chromosome fromsomehow becoming inactivated. This illustrates that although epigeneticsin the form of CpG island methylation and histone acetylation is whatmechanistically turns gene expression on an off for a particular type ofcell, it is epigenetics in the form of the RNA expression network thatkeeps these factors stable whenever necessary for maintaining thedifferentiated phenotype of the cell.

To elaborate upon the point at hand, because turning on one gene cancause it to produce an ncRNA that targets and degrades a different ncRNA(see “RNA interference” above), it is possible to “silence a silencer”,and thereby turn on a downstream gene that would otherwise be silenced.

-   -   “In fact, ncRNAs might be actively switching on and off genes in        an orchestral regulation that governs the fidelity of the cell        and functions in cellular adaptation.” [Morris, 2011]6.6 RTT        Treatments that Increase Histone Acetylation Revisited

In the interest of brevity, only the Tubastatin A, Carnitine,Acetyl-L-Carnitine, Fluvastatin, and G-3 PUFA based treatments will bediscussed in detail here. For more information about how each of theother treatments can increase histone acetylation, consult itsassociated reference listed in Table 2.

Nonetheless, there is a remarkable structural relationship between themolecules associated with many of these treatments. FIG. 1 illustrateshow the acetyl group (FIG. 1A) is present in various molecules ofinterest, either as a group (see the dotted line, which shows where theacetyl group attaches to the rest of the molecule) or as a potentialmetabolite (e.g. with deletion of the contents shown within the dottedboxes). The deletion of the contents of the dotted boxes shown isphysiologically plausible given that the glycerol group gets removedearly during the metabolism of triglycerides (forming 3 fatty acids)(FIG. 1H), Beta oxidation removes pairs of carbons within fatty acids(e.g. FIG. 1E, FIG. 1F, FIG. 1G, FIG. 1H), and Alpha oxidation removesthe odd carbons within fatty acids (FIG. 1G, FIG. 1H). Enzymes areavailable for the formation of Acetyl-CoA from acetyl group donors. Forexample, acetate is readily incorporated into Acetyl-CoA (e.g. by theenzyme acetate thiokinase).

Histone acetyl transferase (HAT) enzymes use Acetyl-CoA as the acetylgroup donor when acetylating histone proteins (i.e. forming acetyllysine (FIG. 1B) at a lysine residue). And histone deacetylase (HDAC)enzymes can remove the acetyl group from a histone's acetyl lysineresidue.

6.6.1 Acetylation is Increased by HDACi, Such as Tubastatin A

The level of histone acetylation is in part dependent upon the relativeactivity of the Histone Acetyl Transferase (HAT) enzymes compared to theactivity of the Histone Deacetylase (HDAC) enzymes. The activity of HDACenzymes can be decreased by HDAC inhibitors (HDACi) such as butyrate orespecially by Tubastatin A.

Experiments using radiolabeled acetate (e.g. [I-¹⁴C]acetate or[methyl-³H]acetate) show that histones can become significantlyacetylated within 15 minutes in response to acetate treatment. This mayeither be due to Acetyl-CoA formed from the radiolabeled acetate beingfollowed by histone acetylation by the HAT enzyme [Boffa, 1978], or bynon-enzymatic histone acetylation [Paik, 1970].

Further experiments show that in the absence of an HDACi (such asbutyrate, 4-carbon long fatty acid), the HDAC enzymes significantlyremove the radiolabeled acetyl groups from purified nuclear histones(FIG. 5, from Boffa, 1978). Interestingly, although less active thanbutyrate, the short chain fatty acids of lengths 3 and 5 also havesignificant HDACi activity (FIG. 6, from Cousens, 1979), resulting insignificantly increased histone acetylation (FIG. 7, from Hinnebusch,2002). These short chain fatty acids could be products of fatty acidcatabolism (or fatty acid synthesis), either by intestinal bacteria orby biological pathways such as β-oxidation or α-oxidation.

-   -   “Inhibition of deacetylases by n-butyrate would seem to be a        general phenomenon among mammalian cells . . . . The relative        lack of specificity for fatty acid chain length . . . coupled        with the noncompetitive nature of inhibition . . . suggest that        butyrate may be acting as a tight binding detergent in        inhibiting these enzymes.” [Cousens, 1979]

In contrast to the high concentration of butyrate that is required forbutyrate to significantly inhibit histone deacetylation (e.g. 5 mM, FIG.5), Tubastatin A is a specific HDACi inhibitor in concentrations as lowas 1 μM (e.g. 5000 times as active a butyrate) (FIG. 8, from Gold,2015).

As shown in FIG. 8, treatment of fibroblast cells from RTT patients(c.806delG, p.Ala40Val in the figure) with 1 μM of Tubastatin Aincreased the expression of acetylated a-tubulin up to that of thenormal control patient (Control in the figure, at 0 μM).

6.6.2 Acetyl-L-Carnitine Increases Acetyl-CoA in Neurons

The cell's nucleus needs a source of Acetyl-CoA for enzymatic histoneacetylation to be possible. Acetyl-CoA is formed in mitochondria, usingeither pyruvate (e.g. from glucose metabolism) or as the product ofβ-Oxidation of fatty acids (FIG. 4, from Madiraju, 2009). Although thisAcetyl-CoA cannot leave the mitochondria, it can transfer its acetylgroup to a carnitine molecule (thanks to the enzyme Carnitine0-AcetylTransferase “CAT”) to form Acetyl-L-Carnitine, which can leavethe mitochondria. The nucleus can take up this Acetyl-L-Carnitine andthen convert it to Acetyl-CoA using the CAT enzyme (running backwards).The increased Acetyl-CoA in the nucleus results in increased HATactivity and increased histone acetylation (FIG. 1 of Madiraju, 2009).Although they used L-carnitine for their experiments (like treatment #1in Table 2), forming the Acetyl-L-Carnitine in mitochondria, treatmentwith Acetyl-L-Carnitine itself (like treatment #11 in Table 2) will havethe same net effect.

In humans, there are three enzymes that can produce Acetyl-CoA fromacetate. Two of these enzymes are located in the mitochondria, andtherefore Carnitine is needed for the Acetyl-CoA to be translocated tothe nucleus (where it can be used to acetylate histones). The thirdenzyme (ACSS2) is located in the cytosol and in the nucleus, and canprovide the nucleus with Acetyl-CoA without requiring mitochondrialactivity. [Comerford, 2014]

6.6.3 Fluvastatin Increases Acetyl-CoA in Neurons

As shown in FIG. 2, statins (including Fluvastatin and Lovastatin)inhibit the activity of the HMG-CoA synthase enzyme, the rate limitingstep in cholesterol biosynthesis. This reduces the flow of Acetyl-CoAinto this pathway, thereby increasing the amount of Acetyl-CoA that isavailable for other purposes, including histone acetylation.

6.6.4 ω-3 Polyunsaturated Fatty Acids increase HDACi Activity

Although the mechanism is unclear, there is ample evidence that ω-3polyunsaturated fatty acids work together with HDACi to unsilenceectopically silenced genes. Most of this research has involved coloncancer, and the HDACi is bacterially produced butyrate.

While not wanting to be bound by any particular theory, the applicantnotes the following possible mechanism(s) of action:

1. ω-3 polyunsaturated fatty acids are known to affect the expression ofhundreds of genes. A comparison of the gene expression profiles betweenrats fed ω-3 vs. ω-6 PUFAs for 10 weeks found 52 genes whose expressionwas significantly higher in the ω-3 fed group and 18 genes whoseexpression was significantly lower [Davidson, 2004]. But what caused thegene expression to change? One answer is that the expression of any onegene depends in part on the expression of other genes. Perhaps thedirect effect of the ω-3 treatment is to increase gene expression (e.g.by histone deacetylation) of some genes, explaining how 52 of the genesincreased their expression (and gene-to-gene interactions explaining howthe other 18 genes decreased their expression). So gene-to-geneinteractions can explain why 18 genes went down, but we still need anexplanation for why the expression of 52 genes went up.

2. There are various known receptors that are known to be activated byω-3 PUFAs (e.g. the peroxisome proliferator-activated receptors (PPARs).This is certainly the most obvious explanation, but it may not be thewhole story.

3. Perhaps ω-3 treatment demethylates the promoter region of genes. Thiscould also explain why the expression of 52 genes increased (and 18decreased from gene-to-gene interactions). It is well established thatthe combination treatment of an HDACi plus a demethylation agent is moreeffective than either treatment alone (see the sections onX-inactivation above). The various studies on the effect of ω-3 andbutyrate on colon cancer cell apoptosis points commonly describe asynergistic effect. But at least one study shows that their combinedeffect is roughly additive (and interestingly, if an ω-6 PUFA is usedinstead of an ω-3 PUFA, the effect is roughly subtractive, perhapsbecause excess ω-6 depletes ω-3 in tissues) (FIG. 9, from Chapkin,2008).

4. Given that the figure from Chapkin shows a nearly additive effect,perhaps the ω-3 PUFA treatment produces HDACi activity. This could bedue to something as simple as short chain fatty acids being one of themetabolites from PUFA catabolism (e.g. from β-oxidation, or α-oxidation,or from some other metabolic pathway).

Without wanting to be bound by any particular theory, the Applicantthinks that there is a combination of receptor activation (e.g. by PPARreceptors), gene-to-gene expression interactions, and HDACi activity dueto short chain fatty acid metabolite(s) that may be formed during themetabolism of ω-3 PUFAs.

In any case, the observed synergy (or at least additivity) between ω-3PUFA and HDACi treatments for increasing gene expression is sufficientto validate that HDACi treatment is beneficial for RTT, in the presenceor absence of ω-3 PUFA treatment.

6.7 Examples of Dietary Compositions for RTT Treatment

Given the variety of beneficial pharmaceutical treatments for RTT shownabove to increase histone acetylation, which one is the best for use inhumans? Or, perhaps the best treatment isn't one of the above.

Given that it is likely that the treatment will need to be administeredfor life, ideally it would have a high degree of safety, goodeffectiveness, and a low degree of side effects.

The &-3 PUFA treatment has a good potential for this, but the idealdosage hasn't been determined yet, and there is a practical limit to howmuch fat should be given to the patient. Also, it isn't clear that ω-3PUFA treatment is efficient at increasing histone acetylation (giventhat the mechanism for this hasn't been completely determined).

Acetate is known to be efficient at rapidly increasing histoneacetylation (see the experiments listed above) [Boffa, 1978], [Paik,1970]. Acetic Acid (vinegar, FIG. 1D) forms acetate in water, becausethe H⁺ of the OH group separates from the molecule (giving vinegar itsacidity).

Vinegar has been used medicinally for a very long time (e.g. inBabylonia in 5,000 BC.), and is still being researched. Surprisingly, noone seems to attribute the medicinal properties of vinegar to itsacetate content. They look for something else: the potassium content ofapple cider vinegar [Orey, 2006; page 20), the flavonoids of red winevinegar [Orey, 2006; page 67], or the polyphenols of basalmic vinegar[Orey, 2006; page 101].

A recent study showed that vinegar intake (750 to 1500 mg of acetate perday) for 12 weeks reduces body weight, BMI, and body fat mass in obeseJapanese subjects, with a dose-dependent response [Kondo, 2009]. Thisdosage is in the range recommended for various medicinal uses, which istypically 1 to 2 tablespoons of apple cider vinegar in a glass of water[Orey, 2006; page 141], which equates to 750 to 1500 mg of acetate forthe normal 5% “acidity” of vinegar.

But vinegar is not for everyone. For one thing, there is the acidcontent, which can cause canker sores, heartburn, tooth damage, bladderpain, flare-ups of interstitial cystitis (bladder infection), and jointdiscomfort [Orey, 2006; pages 211-215]. And some people (most people?)don't like the taste of drinking this much vinegar at once (even whendiluted by a full glass of water).

6.7.1 A Dietary Supplement

But for our purposes, it is not the acidity we are after (or thepotassium, the flavonoids, or the polyphenols). It is the acetyl groupsthat come from the acetate. Instead of having the acetate attached to anH⁺ (which is released when the acetate is dissolved in water), theacetate can be attached to another cation such as sodium, which willrelease a Na⁺ when dissolved in water. Acidity no more.

But for our purposes, there is a better cation than sodium (which somepeople try to avoid). Magnesium is an essential micronutrient which isdeficient in the modern (Western) diet, partially due to magnesiumdepletion of farming soil.

I made some “Magnesium ProAcetyl” capsules by:

1. Start with 250 ml “Heinz Distilled White Vinegar” (5%)

2. While mixing in a Kitchen Aid mixer, add “Freeda Vitamins Inc.”supplement grade Magnesium Carbonate powder until the pH=7.0 (7tablespoons=105 ml=35.7 g)

3. Mix a few minutes longer.

4. Pour the mixture into a non-stick baking tray (with the liquid spreadacross the area of the tray up to the sides of the tray).

5. Place the tray in an “Excalibur” Food Dryer and set the temperatureto 63 degrees C. Run the food dryer overnight.

6. Remove the white, crumbly product from the tray using a wooden spoon.

7. Grind the crumbly product to a powder (use a mortar and pestle) toget 42 g of fine powder.

8. Fill 60 size “00” capsules with the powder (700 mg per capsule, ofwhich ˜600 mg is acetate).

The magnesium carbonate dissolves in water (or the water of vinegar)into Mg2⁺ and carbonate (CO₃ ²⁻). The carbonate reacts with 2 H⁺ to make2 CO₂ (which bubbles out from the mixture), leaving the Mg2′ and 2acetates to form the salt “magnesium acetate” when it dries.

I took these capsules, 2 a day with breakfast for a month, with no illeffects. Basically, they went down without notice.

6.7.2 Another Dietary Supplement

Because USP grade (suitable for use in food, dietary supplements, andcosmetics) calcium acetate is readily available commercially (e.g. fromSpectrum Chemical Mfg. Corp, Gardena Calif., USA), there was no need forme to make my own powder. I put 500 mg each in size “0” “CalciumProAcetyl” capsule, (˜375 mg of acetate per capsule) and took thesecapsules for over a month, with no ill effects. They also go downwithout notice at breakfast.

Similar to the magnesium version, the calcium version dissolves intocalcium ions and acetate ions in water.

6.7.3 A Nutraceutical Food

(To my wife's chagrin) I mixed two capsules worth (1000 mg) of thecalcium acetate powder into a serving of Chicken Bouillabaisse that shemade as one of our favorite dinners [Kenji Lopez-Alt, 2009] to make“ProAcetyl Fortified Chicken Bouillabaisse”. I didn't notice any changein the taste at all.

6.7.4 A Nutraceutical Beverage

I mixed one capsules worth (500 mg) of the calcium acetate powder intoan 8 oz glass of “Simply Orange” juice (Simply Orange Juice Company,Apopka, Fla., USA) and kept it overnight in the refrigerator to make“ProAcetyl Fortified Orange Juice”. It tasted like normal orange juice.

Other beverages that taste normal after the same mixing and agingprocess include “Simply Lemonade” juice (from the same manufacturer asthe orange juice), “V8 100% Vegetable Juice” (Campbell Soup Company),and Canada Dry Ginger Ale” (Dr. Pepper/Seven Up, Inc.).

6.8 Examples of Use

6.8.1 Treatment of RTT

For treating RTT, it is just necessary to administer the appropriateamount of ProAcetyl compositions (capsules, food or beverages) toachieve the desired dosage amount.

Because many RTT patients cannot swallow capsules, either thenutraceutical food or the nutraceutical beverage (or both) should beused, at the choice of the mother.

6.8.2 Dosages

Based upon the medicinal use of vinegar (for treating various ailments)and for the use of Dichloroacetate for treating cancer (see below), theexpectation is that 900 mg of acetate should produce a beneficialresponse. Of course, it would be appropriate to conduct a Phase Iclinical trial to determine both the minimum effective dosage and themaximum tolerable dosage. Because of the very low toxicity of acetate(see below), it is expected that the “therapeutic window” will be verywide.

In the absence of a Phase I clinical trial, the most preferred dosage is1500 mg of acetate per day. A less preferred dosage is 750 mg to 7500 mgof acetate per day. An even less preferred dosage is 375 mg to 15000 mgof acetate per day.

Because the practice in the art is to conduct a Phase I clinical trialto determine the acceptable dosage range for a pharmaceutical treatment,conducting such a clinical trial (or its equivalent) is not regardedwithin the art as undo experimentation.

6.9 Treatment with Acetate Prodrugs is Amazingly Nontoxic

Acetate in the form of acetic acid (FIG. 1D), sodium acetate (FIG. 10A),potassium acetate (FIG. 10B), calcium acetate (FIG. 10E), and glycerintriacetate (FIG. 10F) are all FDA approved food additives that aregenerally recognized as safe. Magnesium acetate is also FDA approved foruse as a dietary supplement. All of these compounds readily produceacetate when consumed by mammals. (Note: the dotted lines in the figuresshow where the acetyl groups connect to the rest of the molecule.)

The most interesting of these (from an oral toxicity standpoint) isglycerin triacetate (also known as triacetin), which has been proposedfor use as a major food source for astronauts during long-term spacemissions (where food needs to be synthesized on the space ship). One ofthese studies was done by the “Committee On Space Research” andpublished as “Current Research on Regenerative Systems” [Shapira, 1968].They conclude that “animals can tolerate up to 20% triacetin” (as apercentage of total diet, replacing conventional carbohydrates).

More extensive toxicity information is provided in the “Final Report onthe Safety Assessment of Triacetin” [CIREP, 2003]:

-   -   “Triacetin was affirmed as a generally recognized as safe (GRAS)        food additive by the Food and Drug Administration (FDA).        Triacetin was not toxic to animals in acute oral or dermal        exposures . . . Triacetin was quickly metabolized into glycerol        and acetic acid and these chemicals were not developmental        toxinsz . . . . Rats were fed for 30 days a diet in which 28.5%        of total calories were supplied by Triacetin. No overt signs of        toxicity were observed.” [CIREP, 2003]

As noted in the report, triacetin was quickly metabolized into glyceroland acetic acid (acetate ions), so the non-toxicity of the acetate fromtriacetin provides evidence that acetate from any other prodrug foracetate (e.g. any compound that metabolizes to form acetate) is alsonon-toxic (i.e. not toxic from the acetate that is formed).

6.10 Increased Histone Acetylation is Good for Your Memory Too

There are various medicinal benefits from treatment with acetyl groups(more about this in other sections below), but it is especiallyinteresting that they improve memory and learning even for people whodon't have Rett syndrome.

Altered histone acetylation is associated with age-dependent memoryimpairment in mice. Treatment with the HDACi drug Vorinostat (SAHA)improves memory for 16 month old mice [Peleg, 2010]. For humans, thedietary supplement Juvenon, which contains acetyl-L-carnitine (an acetylgroup donor) is used to improve the memory performance of old people.Interestingly, Juvenon attributes the benefits of their supplement toacetyl-L-carnitine improving neurotransmitters, readily entering thenervous system, and increasing mitochondrial energy production. They donot attribute any of the benefits to the acetyl group itself.

It has been reported that histone deacetylase 2 [Penny, 2014], histonedeacetylase 3 [Penny, 2014], and histone deacetylase 4 [Sando, 2012]have important roles for the long term potentiation (LTP) that isnecessary for learning and memory. All three of these are responsive totreatments with HDACi drugs, with beneficial results from treatment withbutyrate, valproic acid, Trichostatin A, and SAHA [Penny, 2014].

In particular, HDAC4-dependent signaling appears to be involved in apath supporting synaptic plasticity and memory that has distinctivefeatures when compared to the activity of MeCP2. According to Sando,2012 (references omitted):

-   -   “Similar to HDAC4, the nuclear function of MeCP2 is affected by        the site-specific phosphorylation. None the less, MeCP2        associates with genomic DNA in a histone-like fashion, globally        alters chromatin structure, and impacts virtually thousands of        genes triggering a genome-wide response of chromatin to changes        in neuronal activity. Furthermore, MeCP2 acts as both a        transcriptional repressor and activator, and MeCP2        loss-of-function studies revealed a variety of phenotypes,        including altered neuronal branching, excitatory synapse        numbers, and reduced inhibitory synapse strength. Unlike MeCP2,        HDAC4 appears to interact with sites sparsely distributed across        the genome and influence a relatively restricted pool of genes.”

Without wanting to be restricted to a particular theory, the applicantnotes that there are multiple pathways for memory, learning, andun-learning that operate in parallel (and sequentially) in humans,including: (1) short term potentiation, (2) long term potentiation, (3)memory consolidation (e.g. during sleep), (4) memory revision (e.g. fromrecall, which can modify stored memories), and (5) un-learning (newexperiences updating or replacing what had previously been learned).Perhaps with RTT, one of the learning pathway is lost (unfortunately),but an un-learning pathway that turns on at the same stage ofdevelopment (e.g. for memory consolidation) remains active.

It would make sense for memory consolidation (seeing how a newexperience fits in with past experiences and linking them together) tobe turned-off in infancy, because there hasn't been enough experience tomeaningfully form links. It is known that repeat experiences form thebasis for early learning. But once enough has been learned (from repeatexperiences) a single new experience can be put into context, eitherfitting in (and reinforcing) previous learning or contradicting (andtherefor favoring unlearning) what has previously been learned. This newstage of development (being able to unlearn, based upon a single newexperience) could be the fatal-flaw in the RTT brain.

But for everyone else, the beneficial treatment for RTT (increasedhistone acetylation) could improve our learning and memory, especiallyas we age.

6.11 Reversal of the Age Related Decrease in Histone Acetylation

It is known that the general level of histone acetylation decreases withage. It is also known that inflammation is increased when histoneacetylation is decreases. It is also known that cancer cells have anabnormally low level of histone acetylation. What hasn't beenappreciated in the past is the role of dietary acetyl groups as anessential micronutrient.

The paper “Histone Deacetylase Inhibitors for Treating a Spectrum ofDiseases Not Related to Cancer [Dinarello, 2011] lists a variety ofdiseases and conditions, especially conditions that are caused bychronic inflammation, and this paper in hereby included by reference inits entirety.

6.12 Increased Histone Acetylation Improves Mood and Behavior

-   -   “In the rat, the adult offspring of mothers that exhibit        increased levels of pup licking/grooming (i.e., High LG mothers)        over the first week of life show increased hippocampal        [glucocorticoid receptor] expression, enhanced glucocorticoid        feedback sensitivity, decreased hypothalamic corticotropin        releasing factor expression, and more modest . . . stress        responses compared to animals reared by Low LG mothers . . . .        These studies support an epigenetic mechanism, since the        fostering mother and not the biological genetic mother define        the stress response of its adult offspring . . . . A        comprehensive analysis of the hippocampal transcriptome of the        offspring of High and Low LG mothers revealed differences in a        few hundred genes. This suggests a change in epigenetic        programming in the brain of the offspring as a consequence of        maternal care.” [McGowan, 2008]    -   “This programming by maternal behavior is stable and long        lasting, but . . . is reversible by agents that interfere with        either the methylation or histone deacetylation machinery. Thus        the maternal care model typifies the first principles of        epigenetic programming, which are stability and relative        plasticity.” [McGowan, 2008]    -   . . .    -   “Trichostatin A induces replication-independent demethylation in        cell culture. Trichostatin A induces histone acetylation by        inhibiting HDACs and thus tilting the histone acetylation        towards acetylation. We proposed that this open chromatin        structure induced by the hyperacetylation facilitated the        interaction of demethylases with methylated DNA and thus tilted        the DNA methylation equilibrium toward demethylation (Cervoni        and Szyf, 2001). We therefore addressed the question of whether        the epigenetic programming early in life could be modulated        during adulthood.” [McGowan, 2008].    -   “Trichostatin A [treatment] of adult offspring of Low LG        maternal care increased acetylation, reduced methylation,        activated [the glucocorticoid receptor gene] to levels        indistinguishable from adult offspring of High LG maternal care        and reduced stress response to the levels of offspring of High        LG (Weaver et al., 2004).” [McGowan, 2008]

In humans, childhood stress can affect the stress response and behaviorof an adult. This is likely to involve epigenetic programming, similarto that which occurs experimentally in rats exposed to early-age stress.And this epigenetically programmed excessive stress response is likelyto be reversible by treatments which increase histone acetylation andthereby reduce the methylation of the promoter for the humanglucocorticoid receptor.

This is consistent with the observation that various mood stabilizingand antidepressant drugs are HDAC inhibitors, including valproic acid,lithium chloride, lamotrigine, carbamazepine, oxcarbazepine,levetriacetam, olanzapine, clozapine, clomipramine, citalopram, andduloxetine [Ookubo, 2013]. It seems that these drugs share the samemechanism of action (increased histone acetylation, leading to thereversal of ectopic CpG island methylation) that can be achieved withthe claimed dietary supplements and nutraceuticals.

Therefore, much of the depression and mood abnormalities in modernsociety may be the result in part of a dietary deficiency (the lack ofdietary acetyl groups) that can be readily corrected by theadministration of the claimed dietary supplements and nutraceuticals.6.13 Elimination of Pre-Cancerous Cells (Cancer Stem Cells)

Cancer is considered to be a multitude of diseases. Even a single typeof cancer involves a multitude of mutations, with each individualpatient's cancer having a different mix of mutations. And even a singletumor within an individual patient can have cells with different mixesof mutations, with some mutations in common (e.g. the mutations thatoccurred early in tumorigenesis, and therefore are present in thevarious strains of daughter cells) and other mutations varying due todifferent daughter cells acquiring different mutations as the tumorcells continue to divide.

What all cancers have in common is “genomic instability”, with both amultitude of mutations within the tumor and the ability to continue tomutate rapidly (e.g. to become resistant to cancer treatment). In somecases, resistance to a cancer treatment can develop without requiring anew mutation, because a sub-population of the tumor cells may alreadyhave a resistance to the treatment and now have a selective advantage tofavor their proliferation (as the tumor cells that are sensitive to thetreatment die off). The development of resistance limits theeffectiveness anti-cancer treatments.

Another problem is that most anti-cancer treatments selectively targetrapidly dividing cells, killing the cells when they replicate (tumorcells and hair cells alike), but a tumor may also have cancer stem cellsthat replicate slowly, giving them the ability to survive (perhaps foryears) until the cancer develops again.

6.13.1 Non-Hodgkin's Lymphoma Reversal with Dichloroacetate

-   -   “After being successfully treated with six treatments of Rituxan        plus CHOP (cyclophosphamide, doxorubicin hydrochloride,        vincristine, and prednisolone) regime over a period of three        months in 2007, a positron emission tomography (PET) scan showed        a complete remission of the Non-Hodgkin's Lymphoma. With no        further treatments by August 2008, the PET showed his tumors        returned in the nasopharynx and neck lymph glands which        presented with a low grade fever of 99.8, sweating and fatigue.”        [Flavin, 2010]    -   “The Non-Hodgkin's Lymphoma patient refused conventional        therapy, instead personally obtaining dichloroacetate (DCA) he        began administering 900 mg daily at 10 mg/kg in August 2008,        adding a daily 750 mg of thiamine to protect his nerves from        toxicity. Four months later a PET scan showed complete remission        (see FIG. 2 [of the original article]). He has remained        tumor-free on the continued regime of DCA and thiamine since his        last PET in May 2009. Monthly blood tests are showing that all        of his parameters are normal.” [Flavin, 2010]

Dichloroacetate has been used in medicine for over 30 years as aninvestigational drug to treat severe metabolic disorders as well as thetreatment of congenital lactic acidosis in children. As a medicinal, DCAis generally well tolerated from dosages between 10 mg/kg and 50 mg/kg,although prolonged exposure is associated with peripheral neuropathy.Its activation of the pyruvate dehydrogenase enzyme of the mitochondria(due to its inhibition of pyruvate dehydrogenase kinase) decreasesglycolysis and reactivated glucose oxidation, a favorable approach toameliorate lactic acidosis [Flavin, 2010].

6.13.2 Dichloroacetate Treatment is Beneficial for Many Cancers

The use of dichloroacetate for the treatment of cancer was firstdisclosed in 2007, well before the patent was granted in 2013[Michelakis, 2013]. But due to enthusiastic early and ongoing reportsposted on the internet, patients have been self-treating their cancersfor years (such as reported in the Case Report above [Falvin, 2009]).

This has encouraged various researchers to investigate the effect of DCAon the viability of various types of tumors (either in humans, in animalmodels, or using tumor cells in vitro): (1) non-small lung cancer[Bonnet, 2007]; (2) glioblastoma [Bonnet, 2007; Michelakis, 2010; Shen,2015]; (3) breast cancer [Bonnet, 2007; Sun, 2009; Haugrud, 2014; Gang,2014]; Hong, 2015]; (4) colorectal cancer [Cairns, 2007; Madhok, 2010;Lin, 2014; Delaney, 2014; Ho, 2015]; (5) neuroblastoma [Vella, 2011;Hanberry, 2014]; (6) T cell lymphoma [Kumar, 2012]; (7) C6 glioma [Duan,2013]; (8) prostate cancer [Kailavasan, 2013]; (9) Dalton's lymphoma[Kumar, 2013; Kumar, 2015]; (10) pancreatic cancer [Cairns, 2007;Haugrud, 2014]; (11) melanoma [Abildgaard, 2014]; (12) B-chroniclymphoctic leukemia [Agnoletto, 2014]; (13) recurrent brain tumors[Dunbar, 2014]; (14) medulloblastoma [Di Mango, 2014]; (15) head andneck squamous cell carcinoma [Cernigia, 2015]; and (16) lungadenocarcinoma [Zhou, 2015].

Given that cancer is not a single disease, it may be surprising thatdichloroacetate treatment has been found to be beneficial for so manytypes of cancers. But all types of cancers have something in common. Ifwe could target genomic instability itself, turning the cancer'smultiple mutations against it, we would have a broadly applicable methodfor combating cancer. Could dichloroacetate treatment be doing this? Andif so, how?

In the interest of brevity, I will focus on the first published use ofDCA to inhibit cancer growth [Bonnet, 2007], the patent that resultedfrom this work [Michelakis, 2013]. This work is representative of thestudies that followed, and sets the context for how these studies wereconstructed and how their results were interpreted (so little is lost bynot spending words on them). Then I will discuss the surprising resultsfrom a study which extends this work by showing that DCA also inducesapoptosis in tumor stem cells. [Michelakis, 2010]

6.13.3 DCA Effectively Treats Various Tumor Cell Lines and Rats

-   -   “Although mitochondria are recognized as regulators of apoptosis        [programmed cell death], their importance as targets for cancer        therapy has not been adequately explored or clinically        exploited. In 1930, Warburg suggested that mitochondrial        dysfunction in cancer cells results in a characteristic        phenotype, that is, aerobic glycolysis. Positron emission        tomography (PET) has now confirmed that most malignant tumors        have increased glucose uptake and metabolism . . . . This        suggests that the metabolic phenotype in cancer is due to a        potentially plastic mitochondrial remodeling that results in        suppressed oxidative phosphorylation, enhanced glycolysis, and        suppressed apoptosis.” [Bonnet, 2007]    -   “Whether the metabolism of glucose will end with glycolysis in        the cytoplasm (converting pyruvate to lactate) or continue with        glucose oxidation in the mitochondria is controlled by a        gate-keeping mitochondrial enzyme, pyruvate dehydrogenase (PDH)        . . . . PDH is inhibited by phosphorylation by PDH kinase (PDK)        . . . . In preliminary experiments, we compared several cancer        with normal cell lines and found that cancer cells had more        hyperpolarized mitochondria and were relatively deficient in Kv        channels. If this metabolic-electrical remodeling is an adaptive        response, then its reversal might increase apoptosis and inhibit        cancer growth. We used dichloroacetate (DCA), a small, orally        available small molecule and a well-known inhibitor of PDK.”        [Bonnet, 2007]

In summary, they use DCA to inhibit PDK, which prevents it fromphosphorylizing PDH, causing the tumor cell to switch its energymetabolism from glycolysis in the cytoplasm (characteristic of tumorcells) to glucose oxidation in the mitochondria (characteristic ofnon-tumor cells). By shifting the location of this energy metabolism tothe mitochondria, and thereby decreasing mitochondrial membranehyperpolarization, they think that apoptosis becomes enabled, leading tothe death of the tumor cells. Their experiments with “three human cancercell lines: A549 (non-small-cell lung cancer), MO59K (glioblastoma), andMCF-7 (breast cancer)” and also with rats implanted subcutaneously withA549 tumor cells, appear to support their theory of action for DCAmediated tumor cell apoptosis.

The majority of the data presented in the patent appears to have comefrom this set of experiments with A549, MO59K and MCF-7 tumor cells, andA549 injected rats. But FIGS. 6 and 7 of the patent are new andinteresting. FIG. 6 of the patent clearly shows their model, with DCAentering the mitochondria, and the mitochondria releasing “apoptosisinducing factor” (AIF) which enters the nucleus and also releasingCytochrome C, which activates Caspases, thus triggering apoptosis.

FIG. 7 of the patent supports their conclusion that “DCA's effects arerestricted to mitochondrial pathways.” [Michelakis, 2013]

-   -   “In order to confirm that the effects of DCA are not nonspecific        but are indeed metabolic and regulate apoptosis pathways, a gene        chip and GO analysis of treated and non-treated cells were        performed. We used a “subtraction” strategy to reveal relevant        changes in tumor gene expression that were solely due to DCA.        Studying both the A549 and glioblastoma cell lines (i.e. a very        different tumor than the lung cancer, epithelial versus glial        cells) and focusing on the changes that occurred in a similar        pattern on response to DCA therapy revealed changes in gene        expression due to DCA, rather than idiosyncratic tumor-specific        gene changes.” [Michelakis, 2013]    -   “The genes that were modified by DCA in parallel in A549 (lung)        and M059K (glioblastoma) are listed and their expression levels        were plotted in a heat map (FIG. 7 . . . . Most of these genes        were related to mitochondria and complex I . . . This gene chip        analysis further supports the model described in FIG. 6.”        [Michelakis, 2013]

What is interesting is that although the model for DCA's mechanism ofaction taught by [Bonnet, 2007] and [Michelakis, 2013] as illustrated inFIG. 6 of the patent is well accepted (and repeated in their papers) bythe various studies of DCA listed above, none of the authors haveconsidered the implications (or even the cause) of the gene expressionchanges that are induced by DCA treatment of tumor cells (as shown inFIG. 7 of the patent).

6.13.4 DCA Preferentially Kills Cancer Stem Cells

“When GBM-SCs [glioblastoma multiforme stem cells] were allowed todifferentiate into secondary GBM [glioblastoma multiforme] cell lines,the proportion of cells with GBM-SC markers decreased to a value similarto that of the primary cell lines (˜10%). When allowed to differentiatein the presence of DCA (0.5 mM), however, the proportion of cells withGBM-SC markers was decreased even further to −5%. Indeed, DCA inducedapoptosis in GBM-SC in vitro as well as in GBM primary cell lines.Apoptosis was further increased in GBM-SCs by the combination of DCAplus TMZ [temolozolomide], providing a rationale for combinationtherapy. GBM-SC apoptosis took place in vivo in the post-DCA treatmenttumors. [Michelakis, 2010]

-   -   “Our patients had primary GBMs and the mitochondrial remodeling        was at least partially reversible with DCA, suggesting that is        was not due to irreversible dysfunction. Furthermore, we show        that putative GBM-SC may undergo the same metabolic and        mitochondrial remodeling, but to an enhanced degree, because        GBM-SC had the most hyperpolarized mitochondria both in vivo and        in vitro. Reversal of this mitochondrial remodeling by DCA        induced apoptosis in GBM-SC both in vitro and in vivo. Although        the magnitude of apoptosis induction by DCA is not high        (compared to cytotoxic agents), it is relatively selective,        sparing noncancer cells, and because it involves GBM-SC, may        result in a more sustained clinical effect.” [Michelakis, 2010]

6.13.5 Rethinking the Warburg Effect

The above presented (and commonly accepted) model for the method ofaction of DCA is that by increasing PDH activity (due to PDKinhibition), the Warburg Effect is reversed, allowing the mitochondriato induce apoptosis. But other research shows that it is the tumorsuppressor protein TP53 (Tumor Protein p53, coded by the p53 gene) thatregulates mitochondrial respiration [Matoba, 2006; Ma, 2007] and thatthe restoration of TP53 function leads to the reversal of the Warburgeffect and tumor regression [Ventura, 2007].

The expression of wild type p53 directly causes the expression of theSCO2 gene, producing mRNA for the SCO2 protein (Synthesis of Cytochromec Oxidase 2) [Matoba, 2006]. The SCO2 protein is critical for regulatingthe cytochrome c oxidase (COX) complex, the major site of oxygenutilization in the eukaryotic cell. Disruption of the SCO2 gene in humancancer cells causes the metabolic switch toward glycolysis that isexhibited by TP53-deficient cells [Ma, 2007].

Functional p53 is also needed for the expression of the TIGAR gene(TP53-induced Glycolysis and Apoptosis Regulator) gene [Bensaad, 2006].TIGAR decreases glycolysis by dephosphorylating fructose-2,6-biphosphate(Fru-2,6-P_(z)), an important allosteric effector (+) of the keyglycolytic enzyme 6-phosphofructose-1-kinase (PFK-1) [Ma, 2007].

While TP53 is representative of the various tumor suppressor genes thatare frequently mutated or suppressed in tumor cells, it provides us withan undeniable indicator of how prevalent the inactivation of these genesis in human cancers. If the Warburg effect is a “general property” ofcancers, and it is specifically caused by TP53 inactivation, then thesecancers must all have inactivated TP53.

6.13.6 Cancer Initiation, Progression, and Propagation

The development of cancer (i.e. carcinogenesis) has been shown toinvolve three stages: (1) initiation (a process in which normal cellsare changed so that they are able to form tumors); (2) promotion (aprocess in which existing tumors are stimulated to further grow andchange); and (3) progression (more rapid growth and invasiveness).

A classic animal model of this process involves treating a mouse withDiethylnitrosoamine (DEN) and then later on chronically feeding themouse with Phenobarbital (PB) in the diet. Without the prior initiationwith DEN, feeding PB is not carcinogenic [Kolaja, 1996]. Furthermore,repeated treatments with DEN do not produce a tumor (once is enough, butnot sufficient without a promotor). Clearly, the initiator and thepromotor have different roles in carcinogenesis.

The sequential process of carcinogenesis was studied in detail using awell-established model system of mouse carcinogenesis, the multistageskin tumor progression model [Fraga, 2004].

-   -   “In this model, beginning with normal mouse skin, sequential        topical application of various mutagens, such as [ . . . ], and        tumor promoting agents, such as [ . . . ], generate a spectrum        of different stages of tumorigenesis ranging from premalignant        papilloma to highly metastatic tumors with well-defined genetic        lesions in H-ras or p53. We have examined the aberrant DNA        methylation profile (by candidate gene as well as genomic DNA        and RNA approaches) of all stages of mouse skin tumor        progression, including normal mouse skin; nontumorigenic and        tumorigenic keratinocytes . . . ; benign papilloma cells . . . ;        tumorigenic squamous carcinoma cells representative of different        degrees of differentiation, invasion, and metastasis . . . ; and        highly anaplastic spindle carcinoma lines displaying metastatic        behavior. Aberrantly methylated genes identified by the mouse        study were confirmed in human neoplasms to evaluate the        potential of this system to find clinically relevant genes with        methylation-associated inactivation and also its ability to        serve as a tool to improve understanding of the timing and        hierarchy of epigenetic lesions in human cancer.” [Fraga, 2004]

They tracked the methylation patterns of the CpG islands for 6 genesknown to be hypermethylated in various human cancers. These genes wereBRCA1, MLH1, MGMT, E-cadherin, Snail, and MLT1. They found that thefirst treatment (“initiation”, using DMBA, a well-known tumor initiator)caused the MGMT, Snail, and MLT1 gene promoters to becomehypermethylated, and that these genes were silenced. There was no changein the methylation status of the genes during the next 5 stages of tumorprogression (sequentially treating the cells with various agents knownto cause sequential tumor progression in this mouse model). But themethylation status changed when the cell phenotype transitioned fromepithelial to spindle cell (i.e. spindle cell carcinoma had developed).The E-cadherin CpG island became hypermethylated and the Snail CpGisland lost its hypermethylation.

These results clearly show that treatment with the “initiator” causesgene silencing (by CpG island hypermethylation), but the subsequenttreatments (with “promoting” agents), do not tend to alter themethylation status (but they are probably causing DNA mutations, whichweren't the subject of this study).

Note that when a gene has been silenced, there is no longer a selectiveadvantage for it to mutate, but for genes that have not been silenced,mutation can provide a selective advantage during tumorigenesis. Aftertumor suppressor genes have been silenced, various cells within thetumor will have various mutations from each promoter treatment, and thecells with mutations that provide a growth or survival advantage willbecome the predominant types of cells in the tumor. An advanced tumorwill have a variety of mutated genes, and a set of tumor suppressorgenes that were silenced early on (and are probably not mutated).

Tumor suppressor genes (such as p53, the gene for TP53) are involved indetecting DNA damage, correcting the DNA damage, and preventingreplication of the cell if the DNA damage can't be corrected. TP53 isinvolved with cell cycle control, receiving signals from proteins thatdetect various forms of DNA damage, inducing the genes for proteins thatdo DNA repair, pausing the cell cycle to wait for the DNA to berepaired, and killing the cell (by initiating apoptosis) if the DNAisn't repaired in time.

-   -   “The most common cancer-related change known at the gene level        is p53 mutation . . . . The three most notable features of the        p53 mutation spectra in human cancers are as follows: (i)        transitions at CpG nucleotides [i.e. gene silencing by CpG        island methylation] contribute heavily to the mutation frequency        in many cancers; (ii) a mutation at codon 249 predominates in        HCCs [hepatocellular carcinomas] in individuals from        high-incidence regions [specifically Qidong, China]; and there        is a high frequency of G to T transitions in lung cancer.”        [Hollstein, 1991]

Interestingly, although p53 is the most commonly mutated gene, the mostcommon “mutation” of p53 is its epigenetic silencing (e.g. CpG islandmethylation). p53 can also be inactivated by a signaling pathway (seebelow). The high prevalence of p53 inactivation in cancer explains theWarburg effect, and it also provides a therapeutic target.

The gene mutations associated with Non-Hodgkin's Lymphoma in the brain(also known as Primary Central Nervous System Lymphoma, PCNSL) has beenstudied by at least two groups. Whole Exome Sequencing was performed for9 patients, with 27 genetic mutations showing up in 3 or more patients(FIG. 12) [Vater, 2015]. Interestingly, p53 is not on the list.

Similarly, while the other group found 39 genes that were mutated in twoor more of 9 PCNSL patients, p53 is not on the list (see table below).[Bruno, 2015]

Functional prediction impact (FISM) Genes Chromosome Mutations PatientsNA ≥0.5 ≥0.6 ≥0.7 ≥0.8 ≥0.9 = 1 PIM1 6 32 8 0 8 8 7 6 6 5 IGLL5 22 12 66 0 0 0 0 0 0 MYD88 3 2 5 5 0 0 0 0 0 0 TBL1XR1 3 4 4 0 4 4 4 4 4 3CSMD3 8 4 4 0 4 4 4 3 3 1 CD79B 17 3 3 0 2 2 2 2 2 1 HIST1H2AC 6 8 3 0 33 3 3 1 1 ETV6 12 5 3 0 3 3 2 2 1 1 KLHL14 18 7 2 0 2 2 2 2 2 2 IRF4 6 32 0 2 2 2 2 2 2 PRKCD 3 2 2 0 2 2 2 2 2 2 ABCC8 11 2 2 0 2 2 2 2 2 1ZFHX4 8 2 2 0 2 2 2 2 2 1 SALL3 18 2 2 0 2 2 2 1 1 1 IRF2BP2 1 3 2 0 2 21 1 1 1 CD37 19 2 2 0 2 2 1 1 1 1 OSBPL10 3 7 2 0 2 2 2 2 2 0 EBF1 5 3 20 2 2 2 2 2 0 DST 6 2 2 0 2 2 2 2 1 0 MIF4GD 17 2 2 0 2 2 2 2 1 0HIST1H1D 6 3 2 0 2 2 2 1 1 0 GTB1 12 2 2 0 2 2 2 1 1 0 MEP1B 18 2 2 0 22 2 1 1 0 THBS4 5 2 2 0 2 2 2 1 1 0 ADAMTS5 21 2 2 0 2 2 1 1 1 0HIST1H1E 6 2 2 0 2 1 1 1 1 0 MPEG1 11 3 2 1 1 1 1 1 1 0 OBSCN 1 2 2 0 22 2 2 0 0 C10orf71 10 2 2 0 2 2 2 1 0 0 HMCN1 1 2 2 0 2 2 2 1 0 0 MYH417 2 2 0 2 2 1 1 0 0 TBC1D4 13 2 2 0 2 1 1 1 0 0 SLCA12 6 2 2 0 2 2 1 00 0 ETS1 11 2 2 0 2 2 0 0 0 0 MUC16 19 2 2 2 0 0 0 0 0 0 UNC80 2 2 2 2 00 0 0 0 0 ACTG1 17 1 2 2 0 0 0 0 0 0

Clearly, all of these patients had multiple DNA mutations across a widevariety of genes (indicative of genetic instability), but no p53mutation. If their TP53 protein was operative, the cells with multipleDNA mutations would all have undergone apoptosis long ago. It appearsthat their p53 gene has been silenced (or inactivated by a signalingpathway).

A larger exome sequencing study of 94 tumor samples from patients withDiffuse Large B-cell Lymphoma (DLBCL, similar to PCNSL but not locatedin the brain) found a total of 322 mutated “cancer genes”, with p53mutations in 13 of the patients [13.8%, Supplemental spreadsheet #3 ofZhang, 2013]. This shows that the vast majority of patients did not havep53 mutations (but their p53 was probably silenced or inactivated by asignaling pathway).

Interestingly, DLBCL (and PCNSL) is divided into two subtypes withdifferent prognoses and treatment strategies [Visco, 2012]. Each ofthese types inhibits p53 expression, each by a different pathway.

One type of DLBCL (named “germinal center” or GCB-DLBCL) ischaracterized by the expression of CD10 and of BCL-6. Germinal centerB-cell lymphocytes are “immature” (i.e. only partially differentiated).B-cell lymphocytes are part of the adaptive immune system, with eachB-cell being able to produce a large quantity of a specific antibody(originally produced from randomly rearranging endogenous Ig genes[Cattoretti, 2005]), that successfully targets a specific pathogen. Inorder to produce the specific antibody, the B-cell modifies its own DNA,first by scrambling the codes for “V fragments”, “J fragments” and “Dfragments” that are in every B-cell's DNA, and then cutting and splicingits DNA to write the scrambled pattern into the coding region of a genethat it will express in order to product that specific antibody [Clark,2008]. Because the B-cell (at this stage of development) will be cuttingand splicing its own DNA, it turns off the tumor suppressor genes thatdetect cut DNA. Normally, when the cutting and splicing is completed,the tumor suppressor genes would be enabled again. But the key point isthat, for this cell type, it is normal for these tumor suppressor genesto be turned off (at least for a while). In other words, this isconsistent with the maturation process for a B-cell lymphocyte, andtherefore is not suppressed (e.g. by the cell-type specific pattern ofncRNA expression which maintains the stability of the cellularphenotype).

In effect, the “BCL-6” positive version of DLBCL has aless-differentiated phenotype (which is legitimate for its cell-type)that turns-off p53 (and allows DNA mutations to proceed, withoutinducing apoptosis).

The other type of PCNSL is called “post-germinal center B cells”(post-GBC, also called “Activated B Cell-like” or ABC-DLBCL). This typeof cell is more differentiated and doesn't allow the BCL-6 gene to beexpressed. But it does allow NF-κB (nuclear factorkappa-light-chain-enhancer of activated B cells) to be expressed, whichhas its own pathway for inhibiting p53. NF-κB expression is the cell's“join the army” switch, turning on various aggressive pathways (e.g.generating reactive molecules such as nitric oxide, superoxide, hydrogenperoxide, peroxynitrite, . . . ) to fight nearby pathogens. In order toavoid death by friendly fire, NF-κB protein also inhibits p53(preferring the chance of dying by necrosis to the almost surepossibility of self-damage).

In post-GBC lymphoma, the NF-κB protein is functional (e.g. its gene isnot mutated) and its gene expression is dysregulated, for example by amutation in the gene for a different protein which, through a signalingpathway, turns on the expression of the NF-κB gene. The involvement ofother mutated proteins in NF-κB gene activation was investigated byCompagno, 2009:

-   -   “To investigate whether constitutive NF-κB activation in        ABC-DLBCL represents a primary pathogenic event or reflects the        intrinsic program of the tumor cell of origin, we screened for        mutations the complete coding sequences of 31 NF-κB pathway        genes in 14 samples . . . . This strategy identified a total of        48 sequence changes distributed in 6 different genes, including        the NF-κB regulator TNFAIP/A20 and the positive regulators        CARD11, TNGRSF11A/RANK, TRAF2, TRAF5, and MAP3K7/TAK1. Mutations        were preferentially associated with the ABC-DLBCL phenotype,        where 51.3% of the samples showed alteration in one or more        gene, compared to 22.7% GCB-DLBCL . . . . Analysis of paired        normal DNA, available from 8 samples, indicated the somatic        origin of these events in at least one sample/gene” [In other        words, the mutations were not inherited from a parent.]        [Compagno, 2009]

The gene expression profiles for these two types of PCNSL are incrediblydifferent (see FIG. 3 of Visco, 2012).

An interesting set of experiments has shown that the simple treatmentwith a demethylating agent can eliminate developing tumors even beforethe cancer becomes apparent. In a rat model of esophageal carcinogenesiswhere NBMA (N-nitrosomethylbenzylamine) is used as the initiator andzinc-deficiency is used as the promotor, cancer typically develops in 15weeks. But if the CpG demethylating agent DFMO(a-Difluromethylornithine, also known as Eflornithine) is administeredin drinking water starting at week 5, the cancer is prevented. [Fong,2001]

Immunohistochemical analysis shows that the DFMO treatment restored theexpression of the p16 tumor suppressor gene, which had been silencedbefore the treatment and remained silenced in the control rats. Also,tissue samples from the DFMO treated rats show apoptosis of tumor cellsand tumor shrinkage.

-   -   “The results from this study suggest that effective cancer        prevention by DFMO under the present experimental condition        entails: (a) prompt induction of apoptosis to remove damaged        cells and, thus, reverse esophageal cell proliferation; and (b)        sustained inhibition of cell proliferation to annul the        continued effect of dietary zinc deficiency.” [Fong, 2001]

6.13.7 Alternative Methods for Demethylating CpG Islands

6.13.7.1 5-aza-2′-deoxycytidine (5-azadC, Decitabine, Dacogen)

5-azadC treatment seems to be the most common method used forresearchers for demethylating CpG islands in experiments. And it hasbeen FDA approved as a drug (Decitabine, trade name Dacogen) for use inthe treatment of myelodysplastic syndrome (MDS). It is a nucleosideanalog for cytosine that is mutagenic because it can be incorporated inDNA. When incorporated in DNA, it is a suicidal inhibitor of DNAMethyltransferase enzymes (e.g DNMT1, DNMT3a, DNMT3b) because once theyattach to the 5-azadC in the DNA, they become covalently bound andunavailable for any further DNMT activity. 5-azadC treatment hassignificant side effects.

6.13.7.2 Methotrexate

Methotrexate is an anti-folate drug that blocks the formation ofS-Adenosyl methionine, the methyl donor that is necessary for DNMTactivity. Without DNMT activity, CpG sites become demethylated.Methotrexate is one of the earliest developed chemotherapy drugs (1950)and is still extensively used. However, it has significant side effectsand patients must be monitored (and dosage adjusted) in order to preventcomplications from folate deficiency.

6.13.7.3 Histone Deacetylase Inhibitors Produce Demethylation

Although HDACi (or HAT histone acetyltransfer enzymes, or non-enzymatichistone acetylation) do not have direct demethylase activity, theyinteract with the MBD2 demethylase enzyme, allowing it to demethylateCpG sites more rapidly than the DNMT enzymes remethylate them. Theresult is eventual demethylation (e.g. within days, as in theRNA-mediated demethylation described in section 6.5.3.1 above).

“Valproic acid stimulates active demethylation of ectopically methylated. . . DNA.” [Detich, 2003] For these experiments, cells were harvested48 hours after treatment with valproic acid.

The effect of valproic acid on restoration of p53 expression andsubsequent apoptosis of neuroblastoma cells (after 24 hours) is shown inFIG. 14 (from Condorelli, 2008; VPA is valporic acid, VPM is a VPAanalog, without HDACi activity, Bu is butyrate, MTT is an assay of cellviability). Note the similarity between the valproic acid and butyratecurves (nearly equal response to equal molarity treatment).

Without wanting to be bound to a particular theory, the applicant notesthat it is not clear whether gene expression occurs when the histonesbecome significantly acetylated (loosening the DNA and allowing itstranscription) or waits until the CpG island has become demethylated(perhaps a day later). There are reports of HDACi induced geneexpression without the CpG island being demethylated [Pruitt, 2006].Perhaps a strong initial histone acetylation is sufficient for immediategene expression and the CpG island demethylation (e.g. a day later)serves to re-enforce this. If so, increased histone acetylation mayprove more effective than CpG island demethylation for the restorationof activity of tumor suppressor genes that were previously silenced.

The activity of TP53 as a transcription factor depends upon its abilityto bind to DNA, which in turn depends upon the acetylation of lysineswithin the C-terminal 30 amino acids. Histone deacetylases (e.g. HDAC1,HDAC2, HDAC3, . . . ) reduce the activity of TP53, indicating that it isnot sufficient to merely restore the expression of the p53 gene.

HDACi treatment increases the acetylation of these lysines, restoringthe activity of TP53 [Juan, 2000]. This argues that increased proteinacetylation (e.g. from HDACi treatment) can be preferable todemethylation treatment for restoring TP53 function in tumor cells.

Various forms of mutated p53 which have lost their function as atranscription factor actually retain an ability to induce apoptosisthrough a transcription independent mechanism. The pro-apoptotic proteinBAX is normally bound to the protein Ku70, but acetylated TP53 can causethe BAX to be released, allowing it to migrate to the mitochondria whereit can induce apoptosis [Yamaguchi, 2009]. This additional method ofaction argues that increased protein acetylation (e.g. from HDACitreatment) can be preferable to demethylation treatment for restoringTP53 function in tumor cells, even for cells that have p53 mutations.

6.13.7.4 And Perhaps Dichloroacetate Produces CpG Demethylation

Michalakis reports that “the post-DCA treatment tumors from patients 2to 4 showed increased activity of [. . . TP53] (nuclear translocation),also confirmed by the increased activity and abundance of its downstreamtarget p21. These effects on [TP53] or p21 can also explain . . . andare consistent with the antiproliferative, in addition to theproaptotic, effects of DCA” [Michalakis, 2010].

This report leaves open whether the p53 gene became unsilenced by DCAtreatment (consistent with the Warburg effect, as explained above insection 6.13.5) or existing TP53 protein is translocated to the nucleus.Given that almost all types of tumors have p53 expression inhibited (orp53 mutations, see section 6.13.6 above), it seems extremely unlikelythat there was existing TP53 protein just waiting to enter the nucleus.

However, TP53 is known to require the acetylation of Lys-373 in order tobind to DNA, so perhaps they did actually observe increasedtranslocation to the nucleus (due to increased TP53 acetylation). But ifso, this implies that DCA has HDACi activity (which would explain itsability to reverse the Warburg effect, independent of the ability of DCAto inhibit the PDK enzyme). So the evidence seems to show that DCAtreatment can lead to the activation of previously silenced genes andthe demethylation of CpG islands.

Note: although (in the interest of brevity) this application hasconcentrated upon the tumor suppressor gene p53, there are other tumorsuppressor genes, and other paths to apoptosis, that need to be silenced(or mutated, inhibited by a signaling pathway) for the badly DNA damagedtumor cell to avoid apoptosis. If any of these pathways isepigenetically silenced, but otherwise operational, the reversal of thissilencing will result in the death of the tumor cell. Tumor suppressorgenes which can invoke apoptosis include p53, p73, p21 and p16.

For example, the 13.8% of CNSL lymphoma patients who have p53 mutations(see above) probably still have a functional (but silenced or inhibited)p73, p21 or p16 gene.

6.13.8 What are the Metabolites of Dichloroacetate?

Using radiolabeled starting material, (producing radioactive DCA), itwas determined that up to 50% of DCA is metabolized to carbon dioxide[Fitzsimmons, 2009] (exhaled in breath). The other major metabolitesdetectable in plasma and urine are Glyoxylate and Monochloroacetate(shown in bold in FIG. 15).

As shown in FIG. 15, this is evidence for two major detoxificationpathways, one based upon the zeta class glutathione transferase GST1Z(producing Glyoxylate), and the other based upon an unidentifieddehalogenase (producing Monochloroacetate). The balance between thesetwo pathways depends upon the activity of GST1Z, which varies with ageand with genetic polymorphisms [Shroads, 2008].

Interestingly, one of the metabolites is acetate. It is not surprisingthat this acetate is readily metabolized (producing CO,), given theefficiency of glycerine triacetate as a source of calories (see section6.9 above).

6.13.8 DCA Toxicity

DCA inhibits the activity of the GST1Z enzyme, slowing the clearance ofDCA and shifting the balance of its metabolic pathways towards theproduction of acetone, increasing the concentration ofMonochloroacetate. Furthermore, the GST1Z enzyme is needed for otherdetoxification pathways, including the conversion of Maleylacetone toFumarylacetone. The concentrations of both Maleylacetone andMonochloroacetate (from DCA administration) also rise with age. Thesemolecules may be the cause of DCA induced peripheral neuropathy, themain toxicity of DCA.

DCA treatment also stimulates the activity of at least two enzymes thatrequire thiamine as a cofactor. This can result in thiamine deficiency,which may be the cause of DCA induced peripheral neuropathy [Stacpoole,1990]. This is why the Non-Hodgkins Lymphoma patient took thiamine alongwith his DCA (section 6.13.1 above).

6.13.9 Non-Hodgkin's Lymphoma Reversal with DCA Revisited

We know that for most patients with Non-Hodgkin's Lymphoma their p53tumor suppressor gene is probably not mutated (see section 6.13.6above), but is silenced. The genomic instability that is characteristicof cancer has assured that his tumor cells have multiple mutations, andwould undergo apoptosis if their tumor suppressor genes hadn't beensilenced. We also know that the reversal of this gene silencing (by theHDACi activity of the acetate from the DCA treatment) will allow thesecells do what they should do (i.e. die via apoptosis). So the patient'smiraculous cure (section 6.13.1) isn't a miracle after all.

6.13.10 Alternative “DCA-Like” Treatments for Cancer

The anti-cancer activity of DCA appears to be epigenetic in nature (seesection 6.13.7.4 above). Given that one of the metabolites of DCA isacetate (a well-established HDACi, see section 6.6.2 above), it is clearthat treatment with DCA will have an HDACi effect.

Other sources of acetate will also have this HDACi effect, and can avoidthe toxicity of DCA (e.g. by not inhibiting GST1Z).

For treating cancer with a bioavailable source of acetate, it is justnecessary to administer the appropriate amount of ProAcetyl compositions(capsules, food or beverages) to achieve the desired dosage amount.

Based upon the medicinal use of vinegar (for treating various ailments)and for the use of Dichloroacetate for treating cancer (section 6.13.1),the expectation is that 900 mg of acetate should produce a beneficialresponse. Of course, it would be appropriate to conduct a Phase Iclinical trial to determine both the minimum effective dosage and themaximum tolerable dosage. Because of the very low toxicity of acetate(see below), it is expected that the “therapeutic window” will be verywide.

In the absence of a Phase I clinical trial, the most preferred dosage is1500 mg of acetate per day. A less preferred dosage is 750 mg to 7500 mgof acetate per day. An even less preferred dosage is 375 mg to 15000 mgof acetate per day.

Because the practice in the art is to conduct a Phase I clinical trialto determine the acceptable dosage range for a pharmaceutical treatment,conducting such a clinical trial (or its equivalent) is not regardedwithin the art as undo experimentation. 6.14 Trinucleotide RepeatDisorders 6.14.1 Lovastatin Improves Behavior of Fragile X Patients

Treatment with lovastatin for 12 weeks improves the “Aberrant BehaviorChecklist” score from −50% to −30% in adults and children with Fragile Xsyndrome (FXS, the most common inherited cause of mental disability)[Caku, 2014].

-   -   “In the majority of cases, FXS results from a        [cytosine-guanine-guanine] (CGG) trinucleotide repeat expansion        . . . associated with the methylation of its promoter. This        epigenetic modification leads to transcriptional silencing of        FMR1. Therefore, FXS is characterized by the absence or a        reduced level of expression of the FMR1 protein, FMRP . . . . In        general, the severity of cognitive dysfunction correlates with        the magnitude of FMRP deficit: males being more profoundly        affected than females.” [Caku, 2014]    -   “In normal population this [CGG trinucleotide] repeat is        composed of 5-55 repeats, allowing transcription and translation        of the gene; within this size range the gene is transmitted        stably over generations. When the repeat expands between 56 and        200 (premutation), the gene continues to transcribe (more)        messenger RNA . . . Premutated alleles can expand to over 200        repeats (full mutation) when maternally transmitted, thus        causing transcriptional repression of FMR1 through epigenetic        modifications, namely: cytosine methylation of the expanded        sequence and of the CpG island, deacetylation of histones 3 and        4, demethylation of lysine 4 on histone 3 (H3K4), methylation of        lysine 9 on histone 3 (H3K9) and trimethylation of lysine 27 on        histone 3 (H3K27) . . . preventing transcription and resulting        in the absence of FMRP protein.” [Tabolacci, 2013]

This shows that the trinucleotide repeat ( . . . CGGCGGCGG . . . ),which has a CpG in each of the repeating trinucleotides (shown in boldabove), is acting like a classic CpG island, epigenetically. Indeed, DNAmethyltransferases can recognize and methylate unusual DNA structureslike trinucleotide CGG repeats in vitro [Sandberg, 1997].

Lovastatin treatment, by increasing the concentration of Acetyl-CoA (seesection 3.3.2.2 above), is probably epigenetically unsilencing at leastsome of the FMR1 genes in these patients, allowing the production ofmore FMRP protein.

There is a rare class of FMR1 mutated carriers (with Unmethylated FullMutations, “UFM”) that are phenotypically normal males.

-   -   “The characterization of cell lines derived from these        individuals has revealed that FMR1 promoter DNA is completely        unmethylated (in both the CGG expansion and the FMR1 CpG        island), transcription is increased (as in premutation        carriers), FMRP levels are approximately 30-40% compared to        normal (due to ribosome stalling on the expanded FMR1 mRNA).        [Tabolacci, 2013]

This shows that if we can remove the ectopic methylation of thetrinucleotide repeat, we can convert Fragile X patients to the UFMphenotype (i.e. cure the disease in that patient, without changing theunderlying DNA mutation).

Indeed, in Fragile X embryonic stem cells, where the repeat is stillunmethylated, both FMR1 mRNA and FMRP are made [Baicsi, 2008]. Treatmentwith either the demethylating agent 5-aza-dC or with Splitomycin (SPT, aspecific inhibitor Class III HDAC enzymes), but not Trichostatin A (aless specific HDACi) increased FMR1 mRNA to approximately 20% of normalcontrol cells [FIG. 16 from Baicsi, 2008]. They note that treatment with5-aza-dC is extremely toxic and requires DNA replication to be effective[Baicsi, 2008].

6.14.2 Other Trinucleotide Repeat Disorders

In Fragile XE mental retardation the repeated trinucleotide is CCG(instead of the CGG in Fragile X Syndrome), but this results in a CpGisland just the same (with the CpG sights in bold below): . . .CCGCCGCCG . . .

In Friedreich ataxia, a fatal neuronal disease, the repeat sequenceinvolves two trinucleotides (GAA and TTC) which also produce a CpGisland: . . . GAATTCGAAT . . .

Treatment with the HDACi SAHA (structurally similar to Trichostatin A)or Scriptaid increased FXN gene expression to >20% of normal controlcells [Soragni, 2008].

Another large class of trinucleotide repeat diseases is the“Polyglutamine Diseases”, where the repeating trinucleotide (CAG) codesfor the amino acid glutamine. These diseases include Huntington'sDisease (HD), Dentatorubropallidoluysian atrophy (DRPLA), Spinal andBulbar Muscle Atrophy (SBMA), and Spinocerebellar ataxia Types 1, 2, 3,6, 7, and 17 (ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, and TBP). For all ofthese diseases, the repeat sequence is within the androgen receptor (AR)gene itself: . . . CAGCAGCAG . . .

In this case, the toxicity of the repeat sequence is not due to CpGsites in the DNA. It turns out that the transcription of this sequenceproduces polyglutamine, which forms inclusions which bind to the “CBP”Histone Acetyl Transferase (HAT) proteins and sequesters them. Thiscaused a general hypoacetylation of histone H3, as has been observed inHuntington's Disease. “If sequestration of CBP and other histoneacetylases leads to cell death by decreasing histone acetylation, thenaddition of deacetylase inhibiters would be expected to reduce cellloss. We tested the ability of various deacetylase inhibiters to reducepolyglutamine induced cell death” [McCampbell, 2001].

“In our assay, SAHA [a drug similar to Trichostatin A, TSA] wascomparable to TSA in its ability to reduce cell death . . . We testedtwo other deacetylase inhibitors, sodium butyrate and PBA [phenylbutyricacid]. These compounds, while inducing histone acetylation, have broadereffects on gene expression than TSA. Mariadason et al. [Mariadason,2000] showed that sodium butyrate modifies roughly 10 times as manygenes as TSA in cultured colon cells . . . . The reduction in cell deathcaused by these compounds is not attributable to a decrease in AR [themutant protein] expression levels. Indeed, the expression of [the mutantprotein] is slightly increased by the presence of SAHA, TSA, sodiumbutyrate, and PBA.” [McCampbell, 2001]

“In 2003, there was a report that SAHA added to drinking water of R6/2HD mice, the mouse model for this disease, resulted in a markedimprovement in motor function. Transgenetic mice expression humanHuntington with an expanded CAG/polyglutamine repeat develop aregressive syndrome with many of the characteristics of human Huntingtondisease. Indeed, SHA crosses the blood-brain barrier and increaseshistone acetylation in the brain. The administration of SAHA hasconsistently shown this therapeutic benefit in the mouse model;nevertheless, which HDAC is affected by SAHA to improve motor functionin the R6/2 mouse model remains unclear. 6.15 What Makes AcetateEspecially Efficient as an HDACi?

While now wanting to be bound by any particular theory, the applicantnotes that in E. coli, the enzyme Acetate Kinase (AckA) converts acetateto Acetyl-phosphate (AcP), and the Acetyl-phosphate non-enzymaticallyacetylates a significant percentage of the lysine residues of theproteins in the cell. This occurs when the cell is in a nutrient-poorenvironment, switching the bacteria from its “exponential growth phase”(EP) to its “stationary phase” (SP). [Weinert, 2013]

-   -   ” . . . we compared acetylation between lysine acetylation in        actively growing, exponential phase (EP) cells to acetylation in        stationary phase (SP). Surprisingly, acetylation was        dramatically and globally increased in SP cells . . . .        Increased acetylation was nearly comprehensive (94% of sites        were more than 2-fold elevated), and most sites were more than        an order of magnitude increased in acetylation (median        11.7-fold). The large median increase indicates that more than        half of the sites were less than 8.5% acetylated in EP cells.”        [Weinert, 2013]    -   . . .    -   “In this work we identified more than 8,000 unique acetylation        sites in E. coli and showed that most acetylation occurs at a        low level and is affected by AcP concentration in a uniform        manner . . . . This data established AcP as a critical regulator        of acetylation in bacteria and suggest that AcP acts        non-enzymatically to regulate acetylation levels . . . We found        that [cells without the AckA enzyme] had a median ˜14-fold        higher level of acetylation than [cells that lack the enzyme        that converts Acetyl-phosphate to Acetyl-CoA, decreasing the AcP        level] at thousands of sites.” [Weinert, 2013]

The existence of a non-enzymatic process for histone acetylation mayexplain why there is such a diversity of histone deacetylases (with adiversity of functions) and only a few, relatively nonspecific histoneacetyl transferase enzymes. If histone acetylation is occurringnon-enzymatically (hence, relatively uncontrolled) having thedeacetylase enzymes (which can be part of specific signaling pathways)determine the acetylation/non-acetylation balance of specific siteswould seem to be necessary.

If humans have an analogous pathway from acetate to Acetyl-phosphate,this would explain the ability of acetate from dichloroacetate to bemore effective at inducing the re-expression of formally silenced tumorsuppressor genes than the other HDACi (and demethylases) that have beentested as antitumor agents.

6.16 Revisiting the Warburg Effect (Again)

Without wanting to be bound by a particular theory, the applicant notesthat the cellular switch between an “exponential growth phase” (EP) anda “stationary phase” (SP) is called the “Acetate Switch” [Wolfe, 2005].The ability of cell to switch between these two modes, and itsdependence upon the extracellular acetate concentration, developed firstin eubacterial species (prokaryotes) and has been retained throughevolution, including human cells [Wolfe, 2005].

The “exponential growth phase” is associated with a decrease in proteinacetylation, including decreased histone acetylation. This serves as aglobal signaling mechanism within the cell for these proteins to be in“reproduction” mode. (The converse, increased acetylation in thestationary phase, signals to these proteins that they should be innon-growth mode.)

The Warburg Effect involves a cellular metabolic shift to glycolysis,which prevents pyruvate from entering the Krebs cycle, allowing ATP tobe formed without producing Acetyl-CoA at the same time. This helpsmaintain the low level of acetylation that has been the signal forexponential reproduction, long before tumor suppressor genes (orapoptosis, or even mitochondria) ever existed. It seems that the mainreason for the Warburg Effect is to prevent the formation of Acetyl-CoA,starving the cell of acetyl groups, and putting the tumor cell intoexponential growth mode. And because this mode is controlled by the“acetate switch”, extracellular acetate (which specifically controls theswitch) can switch the tumor out of exponential growth mode.

In summary, acetate is a very efficient source of acetyl groups forenzymatic and non-enzymatic protein acetylation, including histoneacetylation. Dietary sources of acetate can be used to treat a varietyof genetic and epigenetic diseases and disorders. The low toxicity ofdietary sources of acetate such as the FDA approved food additivescalcium acetate, sodium acetate and potassium acetate provides a widetherapeutic window for treatments. Not that these are all simple,ionically bound salts that disassociate in water, yielding only acetateand the non-toxic ions of sodium, calcium and potassium. Similarly, thedietary supplement magnesium acetate yields only acetate and magnesium.

It is especially interesting that DCA preferentially kills cancer stemcells. There is an ongoing problem of disease recurrence after“successful” chemotherapy. Perhaps every patient, after completingchemotherapy or radiation, should also receive ProAcetyl treatment toclear out any cancer stem cells that survived the chemotherapy. This isa low toxicity treatment that can easily be added to conventionalanti-cancer treatments. And the ability to clear cancer stem cells fromthe body could be used periodically (e.g. every year or to) in order toprevent cancers from developing.

What is claimed is:
 1. A method for treating a trinucleotide repeatdisorder in a subject in need thereof, the method comprising orallyadministering to the subject about 375 mg to about 15,000 mg of acetateper day, wherein said acetate is provided as a composition comprisingmagnesium acetate, calcium acetate, or ethylacetate.
 2. The method ofclaim 1, wherein the amount of acetate per day is about 1500 mg to about7500 mg.
 3. The method of claim 1, wherein the amount of acetate per dayis about 750 mg to about 3000 mg.
 4. The method of claim 1, wherein theamount of acetate per day is about 375 mg to about 2000 mg.
 5. Themethod of claim 1, wherein said composition is a nutraceutical.
 6. Themethod of claim 1, wherein said composition is a dietary supplement. 7.The method of claim 6, wherein said dietary supplement is in the form ofa capsule.
 8. The method of claim 1, wherein said composition is apharmaceutical composition.
 9. The method of claim 1, whereinadministration of the composition increases acetylation of histone H3K9and/or histone H3K14 in the subject.
 10. The method of claim 1, whereinthe trinucleotide repeat disorder is Fragile X or Fragile XE.
 11. Themethod of claim 10, wherein the trinucleotide repeat disorder is FragileX, and the administration of the composition increases expression ofFMR1 gene.
 12. The method of claim 1, wherein said trinucleotide repeatdisorder is a polyglutamine disease.
 13. The method of claim 11 whereinsaid polyglutamine disease is Huntington's disease,Dentatorubropallidoluysian atrophy, Spinal and Bulbar Muscle Atrophy,Spinocerebellar ataxia Type 1, Spinocerebellar ataxia Type 2,Spinocerebellar ataxia Type 3, Spinocerebellar ataxia Type 6,Spinocerebellar ataxia Type 7, or Spinocerebellar ataxia Type
 17. 14.The method of claim 1 wherein said trinucleotide repeat disorderinvolves the repeating of a pair of trinucleotides.
 15. The method ofclaim 14 wherein said trinucleotide disorder is Friedreich ataxia.